Treatment of cancer using chimeric antigen receptor and protein kinase a blocker

ABSTRACT

The invention provides compositions and methods for treating diseases associated with expression of a cancer associated antigen as described herein. The invention also relates to chimeric antigen receptor (CAR) specific to a cancer associated antigen as described herein, vectors encoding the same, and recombinant T cells comprising the CARs of the present invention. The invention also includes methods of administering a genetically modified T cell expressing a CAR that comprises an antigen binding domain that binds to a cancer associated antigen as described herein.

This application claims priority to U.S. Ser. No. 62/151,707 filed Apr.23, 2015, the contents of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA66726 awarded bythe National Institute of Health. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates generally to the use of immune effectorcells (e.g., T cells, NK cells) engineered to express a Chimeric AntigenReceptor (CAR) and a RIAD polypeptide to treat a disease associated withexpression of a tumor antigen.

BACKGROUND OF THE INVENTION

Adoptive cell transfer (ACT) therapy with autologous T-cells, especiallywith T-cells transduced with Chimeric Antigen Receptors (CARs), hasshown promise in hematologic cancer trials.

SUMMARY OF THE INVENTION

The present invention pertains, at least in part, to the use of immuneeffector cells (e.g., T cells, NK cells) engineered to express a CARpolypeptide that binds to a tumor antigen as described herein to treatcancer associated with expression of said tumor antigen. In particular,the invention features such cells also including a RIAD or Ezrinpolypeptide as described herein.

CAR-Encoding Nucleic Acids

Accordingly, in one aspect, the invention pertains to an isolatednucleic acid molecule encoding a chimeric antigen receptor (CAR) and/orRIAD or Ezrin polypeptide, wherein the CAR comprises an antigen bindingdomain (e.g., antibody or antibody fragment, TCR or TCR fragment) thatbinds to a tumor antigen as described herein, a transmembrane domain(e.g., a transmembrane domain described herein), and an intracellularsignaling domain (e.g., an intracellular signaling domain describedherein) (e.g., an intracellular signaling domain comprising acostimulatory domain (e.g., a costimulatory domain described herein)and/or a primary signaling domain (e.g., a primary signaling domaindescribed herein). In some embodiments, the tumor antigen is chosen fromone or more of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred toas CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptorvariant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

In some embodiments, tumor antigen bound by the encoded CAR molecule ischosen from one or more of: TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3,CD38, CD44v6, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2,LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2,Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta,TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialicacid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K,OR51E2, TARP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1,MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3,PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR,LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

In certain embodiments, the tumor antigen bound by the encoded CARmolecule is chosen from one or more of: TSHR, CLDN6, GPRC5D, CXORF61,CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2,HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E2.

In some embodiments, the antigen binding domain of the encoded CARmolecule comprises an antibody, an antibody fragment, an scFv, a Fv, aFab, a (Fab′)2, a single domain antibody (SDAB), a VH or VL domain, or acamelid VHH domain.

In some embodiments, the transmembrane domain of the encoded CARmolecule comprises a transmembrane domain chosen from the transmembranedomain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18),ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7,NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46,NKG2D, and/or NKG2C.

In certain embodiments, the encoded transmembrane domain comprises anamino acid sequence of a CD8 transmembrane domain having at least one,two or three modifications but not more than 20, 10 or 5 modificationsof an amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99%identity to an amino acid sequence of SEQ ID NO: 12. In one embodiment,the encoded transmembrane domain comprises the sequence of SEQ ID NO:12.

In other embodiments, the nucleic acid molecule comprises a nucleotidesequence of a CD8 transmembrane domain, e.g., comprising the sequence ofSEQ ID NO: 13, or a sequence with 95-99% identity thereof.

In certain embodiments, the encoded antigen binding domain is connectedto the transmembrane domain by a hinge region. In one embodiment, theencoded hinge region comprises the amino acid sequence of a CD8 hinge,e.g., SEQ ID NO: 2; or the amino acid sequence of an IgG4 hinge, e.g.,SEQ ID NO: 6, or a sequence with 95-99% identity to SEQ ID NO:2 or 6. Inother embodiments, the nucleic acid sequence encoding the hinge regioncomprises a sequence of SEQ ID NO: 3 or SEQ ID NO: 7, corresponding to aCD8 hinge or an IgG4 hinge, respectively, or a sequence with 95-99%identity to SEQ ID NO:3 or 7. In certain embodiments, the chimericantigen receptor comprises a leader region, wherein said leader regionencodes an amino acid sequence comprising SEQ ID NO: 2, or a sequencewith 95-99% identity thereof; or said leader region comprises thenucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence with95-99% identity thereof.

In other embodiments, the nucleic acid molecule encodes an intracellularsignaling domain comprising a sequence encoding a primary signalingdomain and/or a sequence encoding a costimulatory signaling domain. Insome embodiments, the intracellular signaling domain comprises asequence encoding a primary signaling domain. In some embodiments, theintracellular signaling domain comprises a sequence encoding acostimulatory signaling domain. In some embodiments, the intracellularsignaling domain comprises a sequence encoding a primary signalingdomain and a sequence encoding a costimulatory signaling domain.

In certain embodiments, the encoded primary signaling domain comprises afunctional signaling domain of a protein selected from the groupconsisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcRgamma (FCER1G), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa,DAP10, and DAP12.

In one embodiment, the encoded primary signaling domain comprises afunctional signaling domain of CD3 zeta. The encoded CD3 zeta primarysignaling domain can comprise an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:20, or a sequence with 95-99% identity to an amino acid sequence of SEQID NO:18 or SEQ ID NO: 20. In some embodiments, the encoded primarysignaling domain comprises a sequence of SEQ ID NO:18 or SEQ ID NO: 20.In other embodiments, the nucleic acid sequence encoding the primarysignaling domain comprises a sequence of SEQ ID NO:19 or SEQ ID NO: 21,or a sequence with 95-99% identity thereof.

In some embodiments, the encoded intracellular signaling domaincomprises a sequence encoding a costimulatory signaling domain. Forexample, the intracellular signaling domain can comprise a sequenceencoding a primary signaling domain and a sequence encoding acostimulatory signaling domain. In some embodiments, the encodedcostimulatory signaling domain comprises a functional signaling domainof a protein chosen from one or more of CD27, CD28, 4-1BB (CD137), OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically bindswith CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma,IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f,ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX,CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL,DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1,CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, orNKG2D.

In certain embodiments, the encoded costimulatory signaling domaincomprises an amino acid sequence having at least one, two or threemodifications but not more than 20, 10 or 5 modifications of an aminoacid sequence of SEQ ID NO:14 or SEQ ID NO: 16, or a sequence with95-99% identity to an amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16. In one embodiment, the encoded costimulatory signaling domaincomprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In otherembodiments, the nucleic acid sequence encoding the costimulatorysignaling domain comprises a sequence of SEQ ID NO:15 or SEQ ID NO: 17,or a sequence with 95-99% identity thereof.

In other embodiments, the encoded intracellular domain comprises thesequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ IDNO: 18 or SEQ ID NO: 20, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.

In certain embodiments, the nucleic acid sequence encoding theintracellular signaling domain comprises a sequence of SEQ ID NO:15 orSEQ ID NO: 17, or a sequence with 95-99% identity thereof, and asequence of SEQ ID NO:19 or SEQ ID NO:21, or a sequence with 95-99%identity thereof.

In some embodiments, the nucleic acid molecule further comprises aleader sequence. In one embodiment, the leader sequence comprises thesequence of SEQ ID NO: 2.

In certain embodiments, the encoded antigen binding domain has a bindingaffinity KD of 10⁻⁴ M to 10⁻⁸ M.

In one embodiment, the encoded antigen binding domain is an antigenbinding domain described herein, e.g., an antigen binding domaindescribed herein for a target provided above.

In one embodiment, the encoded CAR molecule comprises an antigen bindingdomain that has a binding affinity KD of 10⁻⁴ M to 10⁻⁸ M, e.g., 10⁻⁵ Mto 10⁻⁷ M, e.g., 10⁻⁶ M or 10⁻⁷ M, for the target antigen. In oneembodiment, the antigen binding domain has a binding affinity that is atleast five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or1,000-fold less than a reference antibody, e.g., an antibody describedherein. In one embodiment, the encoded antigen binding domain has abinding affinity at least 5-fold less than a reference antibody (e.g.,an antibody from which the antigen binding domain is derived).

In one aspect, the invention pertains to an isolated nucleic acidmolecule encoding a chimeric antigen receptor (CAR), wherein the CARcomprises an antigen binding domain (e.g., antibody or antibodyfragment, TCR or TCR fragment) that binds to a tumor-supporting antigen(e.g., a tumor-supporting antigen as described herein), a transmembranedomain (e.g., a transmembrane domain described herein), and anintracellular signaling domain (e.g., an intracellular signaling domaindescribed herein) (e.g., an intracellular signaling domain comprising acostimulatory domain (e.g., a costimulatory domain described herein)and/or a primary signaling domain (e.g., a primary signaling domaindescribed herein). In some embodiments, the tumor-supporting antigen isan antigen present on a stromal cell or a myeloid-derived suppressorcell (MDSC).

Vectors

In another aspect, the invention pertains to a vector comprising anucleic acid sequence encoding a CAR and/or RIAD or Ezrin polypeptidedescribed herein. In one embodiment, the vector is chosen from a DNAvector, an RNA vector, a plasmid, a lentivirus vector, adenoviralvector, or a retrovirus vector. In one embodiment, the vector is alentivirus vector.

In an embodiment, the vector comprises a nucleic acid sequence thatencodes a CAR, e.g., a CAR described herein, and a nucleic acid sequencethat encodes an inhibitory molecule comprising: an inhKIR cytoplasmicdomain; a transmembrane domain, e.g., a KIR transmembrane domain; and aninhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIMdomain. In an embodiment the inhibitory molecule is a naturallyoccurring inhKIR, or a sequence sharing at least 50, 60, 70, 80, 85, 90,95, or 99% homology with, or that differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 residues from, a naturally occurring inhKIR.

In an embodiment, the nucleic acid sequence that encodes an inhibitorymolecule comprises: a SLAM family cytoplasmic domain; a transmembranedomain, e.g., a SLAM family transmembrane domain; and an inhibitorcytoplasmic domain, e.g., a SLAM family domain, e.g., an SLAM familyITIM domain. In an embodiment the inhibitory molecule is a naturallyoccurring SLAM family member, or a sequence sharing at least 50, 60, 70,80, 85, 90, 95, or 99% homology with, or that differs by no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues from, a naturallyoccurring SLAM family member.

In one embodiment, the vector further comprises a promoter. In someembodiments, the promoter is chosen from an EF-1 promoter, a CMV IE genepromoter, an EF-1α promoter, an ubiquitin C promoter, or aphosphoglycerate kinase (PGK) promoter. In one embodiment, the promoteris an EF-1 promoter. In one embodiment, the EF-1 promoter comprises asequence of SEQ ID NO: 1.

In one embodiment, the vector is an in vitro transcribed vector, e.g., avector that transcribes RNA of a nucleic acid molecule described herein.In one embodiment, the nucleic acid sequence in the vector furthercomprises a poly(A) tail, e.g., a poly A tail described herein, e.g.,comprising about 150 adenosine bases (SEQ ID NO:33). In one embodiment,the nucleic acid sequence in the vector further comprises a 3′UTR, e.g.,a 3′ UTR described herein, e.g., comprising at least one repeat of a3′UTR derived from human beta-globulin. In one embodiment, the nucleicacid sequence in the vector further comprises promoter, e.g., a T2Apromoter.

CAR Polypeptides

In another aspect, the invention features an isolated CAR polypeptidemolecule comprising an antigen binding domain, a transmembrane domain,and an intracellular signaling domain, wherein said antigen bindingdomain binds to a tumor antigen chosen from one or more of: CD19, CD123,CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA,Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT,IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24,PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1,EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, IGF-I receptor, CAIX,LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5,HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6,TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1,GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP,WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17,XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,legumain, HPV E6, E7, intestinal carboxyl esterase, mut hsp70-2, CD79a,CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75,GPC3, FCRL5, and IGLL1.

In some embodiments, the antigen binding domain of the CAR polypeptidemolecule binds to a tumor antigen chosen from one or more of: TSHR,CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13Ra2,IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1,EGFR, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D,CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML,sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG(TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1,MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2,CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2,LY75, GPC3, FCRL5, and IGLL1.

In some embodiments, the antigen binding domain of the CAR polypeptidemolecule binds to a tumor antigen chosen from one or more of: TSHR,CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1,GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E2.

In some embodiments, the antigen binding domain of the CAR polypeptidemolecule comprises an antibody, an antibody fragment, an scFv, a Fv, aFab, a (Fab′)2, a single domain antibody (SDAB), a VH or VL domain, or acamelid VHH domain.

In some embodiments, the antigen binding domain of the CAR polypeptidemolecule comprises a transmembrane domain of a protein chosen from analpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45,CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS(CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103,ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D,and/or NKG2C.

In other embodiments, the transmembrane domain of the CAR polypeptidemolecule comprises an amino acid sequence having at least one, two orthree modifications but not more than 20, 10 or 5 modifications of anamino acid sequence of a CD8 transmembrane domain, e.g., SEQ ID NO: 12,or a sequence with 95-99% identity to an amino acid sequence of SEQ IDNO: 12. In one embodiment, the transmembrane domain comprises a sequenceof SEQ ID NO: 12.

In other embodiments, the antigen binding domain of the CAR polypeptidemolecule is connected to the transmembrane domain by a hinge region. Inone embodiment, the encoded hinge region comprises the amino acidsequence of a CD8 hinge, e.g., SEQ ID NO: 2, or the amino acid sequenceof an IgG4 hinge, e.g., SEQ ID NO: 6, or a sequence with 95-99% identitythereof.

In other embodiments, the intracellular signaling domain of the CARpolypeptide molecule comprises a primary signaling domain and/or acostimulatory signaling domain. In other embodiments, the intracellularsignaling domain of the CAR polypeptide molecule comprises a primarysignaling domain. In other embodiments, the intracellular signalingdomain of the CAR polypeptide molecule comprises a costimulatorysignaling domain. In yet other embodiments, the intracellular signalingdomain of the CAR polypeptide molecule comprises a primary signalingdomain and a costimulatory signaling domain.

In other embodiments, the primary signaling domain of the CARpolypeptide molecule comprises a functional signaling domain of aprotein selected from the group consisting of CD3 zeta, CD3 gamma, CD3delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc EpsilonR1b), CD79a, CD79b, Fcgamma RIIa, DAP10, and DAP12. In one embodiment,the primary signaling domain comprises a functional signaling domain ofCD3 zeta. The CD3 zeta primary signaling domain can comprise an aminoacid sequence having at least one, two or three modifications but notmore than 20, 10 or 5 modifications of an amino acid sequence of SEQ IDNO: 18 or SEQ ID NO: 20, or a sequence with 95-99% identity to an aminoacid sequence of SEQ ID NO:18 or SEQ ID NO: 20. In some embodiments, theprimary signaling domain of the CAR polypeptide molecule comprises asequence of SEQ ID NO:18 or SEQ ID NO: 20.

In some embodiments, the intracellular signaling domain of the CARpolypeptide molecule comprises a sequence encoding a costimulatorysignaling domain. For example, the intracellular signaling domain cancomprise a sequence encoding a primary signaling domain and a sequenceencoding a costimulatory signaling domain. In some embodiments, theencoded costimulatory signaling domain comprises a functional signalingdomain of a protein chosen from one or more of CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46,or NKG2D.

In certain embodiments, the costimulatory signaling domain of the CARpolypeptide molecule comprises an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16, or a sequence with 95-99% identity to an amino acid sequence of SEQID NO:14 or SEQ ID NO: 16. In one embodiment, the encoded costimulatorysignaling domain comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 16.In other embodiments, the intracellular domain of the CAR polypeptidemolecule comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, andthe sequence of SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequencescomprising the intracellular signaling domain are expressed in the sameframe and as a single polypeptide chain.

In some embodiments, the CAR polypeptide molecule further comprises aleader sequence. In one embodiment, the leader sequence comprises thesequence of SEQ ID NO: 2.

In certain embodiments, the antigen binding domain of the CARpolypeptide molecule has a binding affinity KD of 10⁻⁴ M to 10⁻⁸M. Inone embodiment, the antigen binding domain is an antigen binding domaindescribed herein, e.g., an antigen binding domain described herein for atarget provided above. In one embodiment, the CAR molecule comprises anantigen binding domain that has a binding affinity KD of 10⁻⁴ M to 10⁻⁸M, e.g., 10⁻⁵ M to 10⁻⁷ M, e.g., 10⁻⁶ M or 10⁻⁷ M, for the targetantigen. In one embodiment, the antigen binding domain has a bindingaffinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold,100-fold or 1,000-fold less than a reference antibody, e.g., an antibodydescribed herein. In one embodiment, the encoded antigen binding domainhas a binding affinity at least 5-fold less than a reference antibody(e.g., an antibody from which the antigen binding domain is derived).

In another aspect, the invention features an isolated CAR polypeptidemolecule comprising an antigen binding domain, a transmembrane domain,and an intracellular signaling domain, wherein said antigen bindingdomain binds to a tumor-supporting antigen (e.g., a tumor-supportingantigen as described herein). In some embodiments, the tumor-supportingantigen is an antigen present on a stromal cell or a myeloid-derivedsuppressor cell (MDSC).

CAR-Expressing Cells

In another aspect, the invention pertains to a cell, e.g., an immuneeffector cell, (e.g., a population of cells, e.g., a population ofimmune effector cells) comprising a nucleic acid molecule, a CARpolypeptide molecule, or a vector as described herein.

In one embodiment, the cell is a human T cell. In one embodiment, thecell is a cell described herein, e.g., a human T cell, e.g., a human Tcell described herein; or a human NK cell, e.g., a human NK celldescribed herein. In one embodiment, the human T cell is a CD8+ T cell.In one embodiment, the cell is a T cell and the T cell is diacylglycerolkinase (DGK) deficient. In one embodiment, the cell is a T cell and theT cell is Ikaros deficient. In one embodiment, the cell is a T cell andthe T cell is both DGK and Ikaros deficient.

In another embodiment, a CAR-expressing immune effector cell describedherein can further express another agent, e.g., an agent which enhancesthe activity of a CAR-expressing cell. For example, in one embodiment,the agent can be an agent which inhibits an inhibitory molecule.Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3,CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4 and TGFR beta, e.g., as described herein. Inone embodiment, the agent that inhibits an inhibitory molecule comprisesa first polypeptide, e.g., an inhibitory molecule, associated with asecond polypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRbeta, or a fragment of any of these, and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD-1 or a fragment thereof, and a second polypeptide ofan intracellular signaling domain described herein (e.g., a CD28, CD27,OX40 or 4-IBB signaling domain described herein and/or a CD3 zetasignaling domain described herein).

In one embodiment, the CAR-expressing immune effector cell describedherein can further comprise a second CAR, e.g., a second CAR thatincludes a different antigen binding domain, e.g., to the same target(e.g., a target described above) or a different target. In oneembodiment, the second CAR includes an antigen binding domain to atarget expressed on the same cancer cell type as the target of the firstCAR. In one embodiment, the CAR-expressing immune effector cellcomprises a first CAR that targets a first antigen and includes anintracellular signaling domain having a costimulatory signaling domainbut not a primary signaling domain, and a second CAR that targets asecond, different, antigen and includes an intracellular signalingdomain having a primary signaling domain but not a costimulatorysignaling domain.

While not wishing to be bound by theory, placement of a costimulatorysignaling domain, e.g., 4-1BB, CD28, CD27 or OX-40, onto the first CAR,and the primary signaling domain, e.g., CD3 zeta, on the second CAR canlimit the CAR activity to cells where both targets are expressed. In oneembodiment, the CAR expressing immune effector cell comprises a firstCAR that includes an antigen binding domain that targets, e.g., a targetdescribed above, a transmembrane domain and a costimulatory domain and asecond CAR that targets an antigen other than antigen targeted by thefirst CAR (e.g., an antigen expressed on the same cancer cell type asthe first target) and includes an antigen binding domain, atransmembrane domain and a primary signaling domain. In anotherembodiment, the CAR expressing immune effector cell comprises a firstCAR that includes an antigen binding domain that targets, e.g., a targetdescribed above, a transmembrane domain and a primary signaling domainand a second CAR that targets an antigen other than antigen targeted bythe first CAR (e.g., an antigen expressed on the same cancer cell typeas the first target) and includes an antigen binding domain to theantigen, a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing immune effector cell comprises aCAR described herein, e.g., a CAR to a target described above, aninhibitory CAR, and/or a RIAD or Ezrin polypeptide. In one embodiment,the inhibitory CAR comprises an antigen binding domain that binds anantigen found on normal cells but not cancer cells, e.g., normal cellsthat also express the target. In one embodiment, the inhibitory CARcomprises the antigen binding domain, a transmembrane domain and anintracellular domain of an inhibitory molecule. For example, theintracellular domain of the inhibitory CAR can be an intracellulardomain of PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRbeta.

In one embodiment, an immune effector cell (e.g., T cell, NK cell)comprises a first CAR comprising an antigen binding domain that binds toa tumor antigen as described herein, and a second CAR comprising a PD1extracellular domain or a fragment thereof.

In one embodiment, the cell further comprises an inhibitory moleculecomprising: an inhKIR cytoplasmic domain; a transmembrane domain, e.g.,a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g.,an ITIM domain, e.g., an inhKIR ITIM domain. In an embodiment theinhibitory molecule is a naturally occurring inhKIR, or a sequencesharing at least 50, 60, 70, 80, 85, 90, 95, or 99% homology with, orthat differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20residues from, a naturally occurring inhKIR.

In one embodiment, the cell further comprises an inhibitory moleculecomprising: a SLAM family cytoplasmic domain; a transmembrane domain,e.g., a SLAM family transmembrane domain; and an inhibitor cytoplasmicdomain, e.g., a SLAM family domain, e.g., an SLAM family ITIM domain. Inan embodiment the inhibitory molecule is a naturally occurring SLAMfamily member, or a sequence sharing at least 50, 60, 70, 80, 85, 90,95, or 99% homology with, or that differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 residues from, a naturally occurring SLAMfamily member.

In one embodiment, the second CAR in the cell is an inhibitory CAR,wherein the inhibitory CAR comprises an antigen binding domain, atransmembrane domain, and an intracellular domain of an inhibitorymolecule. The inhibitory molecule can be chosen from one or more of:PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5. In one embodiment, thesecond CAR molecule comprises the extracellular domain of PD1 or afragment thereof.

In embodiments, the second CAR molecule in the cell further comprises anintracellular signaling domain comprising a primary signaling domainand/or an intracellular signaling domain.

In other embodiments, the intracellular signaling domain in the cellcomprises a primary signaling domain comprising the functional domain ofCD3 zeta and a costimulatory signaling domain comprising the functionaldomain of 4-1BB.

In one embodiment, the second CAR molecule in the cell comprises theamino acid sequence of SEQ ID NO: 26.

In certain embodiments, the antigen binding domain of the first CARmolecule comprises a scFv and the antigen binding domain of the secondCAR molecule does not comprise a scFv. For example, the antigen bindingdomain of the first CAR molecule comprises a scFv and the antigenbinding domain of the second CAR molecule comprises a camelid VHHdomain.

Methods of Treatment/Combination Therapies

In another aspect, the present invention provides a method comprisingadministering a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein, or a cell comprising one or more nucleic acidsencoding a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein. In one embodiment, the subject has a disorderdescribed herein, e.g., the subject has cancer, e.g., the subject has acancer which expresses a target antigen described herein. In oneembodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating asubject having a disease associated with expression of a cancerassociated antigen as described herein comprising administering to thesubject an effective amount of a cell comprising a CAR molecule and/orRIAD or Ezrin polypeptide, e.g., as described herein.

In yet another aspect, the invention features a method of treating asubject having a disease associated with expression of a tumor antigen,comprising administering to the subject an effective amount of a cell,e.g., an immune effector cell (e.g., a population of immune effectorcells) comprising a CAR molecule, wherein the CAR molecule comprises anantigen binding domain, a transmembrane domain, and an intracellulardomain, said intracellular domain comprises a costimulatory domainand/or a primary signaling domain, wherein said antigen binding domainbinds to the tumor antigen associated with the disease, e.g. a tumorantigen as described herein.

In a related aspect, the invention features a method of treating asubject having a disease associated with expression of a tumor antigen.The method comprises administering to the subject an effective amount ofa cell, e.g., an immune effector cell (e.g., a population of immuneeffector cells) comprising a CAR molecule, in combination with an agentthat increases the efficacy of the immune cell, wherein:

the CAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain comprising a costimulatory domainand/or a primary signaling domain, wherein said antigen binding domainbinds to the tumor antigen associated with the disease, e.g. a tumorantigen as disclosed herein; and

the agent that increases the efficacy of the immune cell is chosen fromone or more of:

a protein phosphatase inhibitor;

a kinase inhibitor;

a cytokine;

an inhibitor of an immune inhibitory molecule; or

an agent that decreases the level or activity of a T_(REG) cell.

In a related aspect, the invention features a method of treating asubject having a disease associated with expression of a tumor antigen,comprising administering to the subject an effective amount of a cell,e.g., an immune effector cell (e.g., a population of immune effectorcells) comprising a CAR molecule, wherein:

the CAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain comprising a costimulatory domainand/or a primary signaling domain, wherein said antigen binding domainbinds to the tumor antigen associated with the disease, e.g., a tumorantigen as disclosed herein; and

the antigen binding domain of the CAR molecule has a binding affinity atleast 5-fold less than an antibody from which the antigen binding domainis derived.

In another aspect, the invention features a composition comprising animmune effector cell (e.g., a population of immune effector cells)comprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein for use in the treatment of a subject having a diseaseassociated with expression of a tumor antigen, e.g., a disorder asdescribed herein.

In certain embodiments of any of the aforesaid methods or uses, thedisease associated with a tumor antigen, e.g., a tumor antigen describedherein, is selected from a proliferative disease such as a cancer ormalignancy or a precancerous condition such as a myelodysplasia, amyelodysplastic syndrome or a preleukemia, or is a non-cancer relatedindication associated with expression of a tumor antigen describedherein. In one embodiment, the disease is a cancer described herein,e.g., a cancer described herein as being associated with a targetdescribed herein. In one embodiment, the disease is a hematologiccancer. In one embodiment, the hematologic cancer is leukemia. In oneembodiment, the cancer is selected from the group consisting of one ormore acute leukemias including but not limited to B-cell acute lymphoidleukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acutelymphoid leukemia (ALL); one or more chronic leukemias including but notlimited to chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL); additional hematologic cancers or hematologic conditionsincluding, but not limited to B cell prolymphocytic leukemia, blasticplasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse largeB cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-or a large cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma,multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and to disease associated with expression of a tumor antigen describedherein include, but not limited to, atypical and/or non-classicalcancers, malignancies, precancerous conditions or proliferative diseasesexpressing a tumor antigen as described herein; and any combinationthereof. In another embodiment, the disease associated with a tumorantigen described herein is a solid tumor.

In certain embodiments of any of the aforesaid methods or uses, thetumor antigen associated with the disease is chosen from one or more of:CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII,GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA,EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2,LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP,IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, FucosylGM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta,TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK,Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3,GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53, p53 mutant, prostein, survivin andtelomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS,SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerasereverse transcriptase, RU1, RU2, legumain, HPV E6, E7, intestinalcarboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2,CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

In other embodiments of any of the aforesaid methods or uses, the tumorantigen associated with the disease is chosen from one or more of: TSHR,TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT,IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta,SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1, sLe, GM3,TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R,CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1,GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP,WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocationbreakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgenreceptor, Cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1,LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF,CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

In other embodiments of any of the aforesaid methods or uses, the tumorantigen associated with the disease is chosen from one or more of: TSHR,CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1,GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, and OR51E2.

In certain embodiments, the methods or uses are carried out incombination with an agent that increases the efficacy of the immuneeffector cell, e.g., an agent as described herein.

In any of the aforesaid methods or uses, the disease associated withexpression of the tumor antigen is selected from the group consisting ofa proliferative disease, a precancerous condition, a cancer, and anon-cancer related indication associated with expression of the tumorantigen.

The cancer can be a hematologic cancer, e.g., a cancer chosen from oneor more of chronic lymphocytic leukemia (CLL), acute leukemias, acutelymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cellacute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), Bcell prolymphocytic leukemia, blastic plasmacytoid dendritic cellneoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicularlymphoma, hairy cell leukemia, small cell- or a large cell-follicularlymphoma, malignant lymphoproliferative conditions, MALT lymphoma,mantle cell lymphoma, marginal zone lymphoma, multiple myeloma,myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma,Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cellneoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.

The cancer can also be chosen from colon cancer, rectal cancer,renal-cell carcinoma, liver cancer, non-small cell carcinoma of thelung, cancer of the small intestine, cancer of the esophagus, melanoma,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular malignant melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, solid tumors ofchildhood, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, T-cell lymphoma, environmentally inducedcancers, combinations of said cancers, and metastatic lesions of saidcancers.

In certain embodiments of the methods or uses described herein, the CARmolecule is administered in combination with an agent that increases theefficacy of the immune effector cell, e.g., one or more of a proteinphosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor ofan immune inhibitory molecule; or an agent that decreases the level oractivity of a T_(REG) cell.

In certain embodiments of the methods or uses described herein, theprotein phosphatase inhibitor is a SHP-1 inhibitor and/or an SHP-2inhibitor.

In other embodiments of the methods or uses described herein, kinaseinhibitor is chosen from one or more of a CDK4 inhibitor, a CDK4/6inhibitor (e.g., palbociclib), a BTK inhibitor (e.g., ibrutinib orRN-486), an mTOR inhibitor (e.g., rapamycin or everolimus (RAD001)), anMNK inhibitor, or a dual P13K/mTOR inhibitor. In one embodiment, the BTKinhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK).

In other embodiments of the methods or uses described herein, the agentthat inhibits the immune inhibitory molecule comprises an antibody orantibody fragment, an inhibitory nucleic acid, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN) that inhibits the expression of the inhibitorymolecule.

In other embodiments of the methods or uses described herein, the agentthat decreases the level or activity of the T_(REG) cells is chosen fromcyclophosphamide, anti-GITR antibody, CD25-depletion, or a combinationthereof.

In certain embodiments of the methods or uses described herein, theimmune inhibitory molecule is selected from the group consisting of PD1,PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFRbeta, CEACAM-1, CEACAM-3, and CEACAM-5.

In other embodiments, the agent that inhibits the inhibitory moleculecomprises a first polypeptide comprising an inhibitory molecule or afragment thereof and a second polypeptide that provides a positivesignal to the cell, and wherein the first and second polypeptides areexpressed on the CAR-containing immune cells, wherein (i) the firstpolypeptide comprises PD1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, and CEACAM-5 ora fragment thereof; and/or (ii) the second polypeptide comprises anintracellular signaling domain comprising a primary signaling domainand/or a costimulatory signaling domain. In one embodiment, the primarysignaling domain comprises a functional domain of CD3 zeta; and/or thecostimulatory signaling domain comprises a functional domain of aprotein selected from 41BB, CD27 and CD28.

In other embodiments, cytokine is chosen from IL-7, IL-15 or IL-21, orboth.

In other embodiments, the immune effector cell comprising the CARmolecule and a second, e.g., any of the combination therapies disclosedherein (e.g., the agent that that increases the efficacy of the immuneeffector cell) are administered substantially simultaneously orsequentially.

In other embodiments, the immune cell comprising the CAR molecule isadministered in combination with a molecule that targets GITR and/ormodulates GITR function. In certain embodiments, the molecule targetingGITR and/or modulating GITR function is administered prior to theCAR-expressing cell or population of cells, or prior to apheresis.

In one embodiment, lymphocyte infusion, for example allogeneiclymphocyte infusion, is used in the treatment of the cancer, wherein thelymphocyte infusion comprises at least one CAR-expressing cell of thepresent invention. In one embodiment, autologous lymphocyte infusion isused in the treatment of the cancer, wherein the autologous lymphocyteinfusion comprises at least one CAR-expressing cell described herein.

In one embodiment, the cell is a T cell and the T cell is diacylglycerolkinase (DGK) deficient. In one embodiment, the cell is a T cell and theT cell is Ikaros deficient. In one embodiment, the cell is a T cell andthe T cell is both DGK and Ikaros deficient.

In one embodiment, the method includes administering a cell expressingthe CAR molecule and/or RIAD or Ezrin polypeptide, e.g., as describedherein, in combination with an agent which enhances the activity of aCAR-expressing cell, wherein the agent is a cytokine, e.g., IL-7, IL-15,IL-21, or a combination thereof. The cytokine can be delivered incombination with, e.g., simultaneously or shortly after, administrationof the CAR-expressing cell. Alternatively, the cytokine can be deliveredafter a prolonged period of time after administration of theCAR-expressing cell, e.g., after assessment of the subject's response tothe CAR-expressing cell. In one embodiment the cytokine is administeredto the subject simultaneously (e.g., administered on the same day) withor shortly after administration (e.g., administered 1 day, 2 days, 3days, 4 days, 5 days, 6 days, or 7 days after administration) of thecell or population of cells of any of claims 61-80. In otherembodiments, the cytokine is administered to the subject after aprolonged period of time (e.g., e.g., at least 2 weeks, 3 weeks, 4weeks, 6 weeks, 8 weeks, 10 weeks, or more) after administration of thecell or population of cells of any of claims 61-80, or after assessmentof the subject's response to the cell.

In other embodiments, the cells expressing a CAR molecule areadministered in combination with an agent that ameliorates one or moreside effects associated with administration of a cell expressing a CARmolecule. Side effects associated with the CAR-expressing cell can bechosen from cytokine release syndrome (CRS) or hemophagocyticlymphohistiocytosis (HLH).

In embodiments of any of the aforesaid methods or uses, the cellsexpressing the CAR molecule are administered in combination with anagent that treats the disease associated with expression of the tumorantigen, e.g., any of the second or third therapies disclosed herein.Additional exemplary combinations include one or more of the following.

In another embodiment, the cell expressing the CAR molecule, e.g., asdescribed herein, can be administered in combination with another agent,e.g., a kinase inhibitor and/or checkpoint inhibitor described herein.In an embodiment, a cell expressing the CAR molecule can further expressanother agent, e.g., an agent which enhances the activity of aCAR-expressing cell.

For example, in one embodiment, the agent that enhances the activity ofa CAR-expressing cell can be an agent which inhibits an inhibitorymolecule (e.g., an immune inhibitor molecule). Examples of inhibitorymolecules include PD1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4and TGFR beta.

In one embodiment, the agent that inhibits the inhibitory molecule is aninhibitory nucleic acid is a dsRNA, a siRNA, or a shRNA. In embodiments,the inhibitory nucleic acid is linked to the nucleic acid that encodes acomponent of the CAR molecule. For example, the inhibitory molecule canbe expressed on the CAR-expressing cell.

In another embodiment, the agent which inhibits an inhibitory molecule,e.g., is a molecule described herein, e.g., an agent that comprises afirst polypeptide, e.g., an inhibitory molecule, associated with asecond polypeptide that provides a positive signal to the cell, e.g., anintracellular signaling domain described herein. In one embodiment, theagent comprises a first polypeptide, e.g., of an inhibitory moleculesuch as PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFRbeta, or a fragment of any of these (e.g., at least a portion of theextracellular domain of any of these), and a second polypeptide which isan intracellular signaling domain described herein (e.g., comprising acostimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as describedherein) and/or a primary signaling domain (e.g., a CD3 zeta signalingdomain described herein). In one embodiment, the agent comprises a firstpolypeptide of PD1 or a fragment thereof (e.g., at least a portion ofthe extracellular domain of PD1), and a second polypeptide of anintracellular signaling domain described herein (e.g., a CD28 signalingdomain described herein and/or a CD3 zeta signaling domain describedherein).

In one embodiment, the CAR-expressing immune effector cell of thepresent invention, e.g., T cell or NK cell, is administered to a subjectthat has received a previous stem cell transplantation, e.g., autologousstem cell transplantation.

In one embodiment, the CAR-expressing immune effector cell of thepresent invention, e.g., T cell or NK cells, is administered to asubject that has received a previous dose of melphalan.

In one embodiment, the cell expressing a CAR molecule, e.g., a CARmolecule described herein, is administered in combination with an agentthat increases the efficacy of a cell expressing a CAR molecule, e.g.,an agent described herein.

In one embodiment, the cells expressing a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein, are administered incombination with a low, immune enhancing dose of an mTOR inhibitor.While not wishing to be bound by theory, it is believed that treatmentwith a low, immune enhancing, dose (e.g., a dose that is insufficient tocompletely suppress the immune system but sufficient to improve immunefunction) is accompanied by a decrease in PD-1 positive T cells or anincrease in PD-1 negative cells. PD-1 positive T cells, but not PD-1negative T cells, can be exhausted by engagement with cells whichexpress a PD-1 ligand, e.g., PD-L1 or PD-L2.

In an embodiment this approach can be used to optimize the performanceof CAR cells described herein in the subject. While not wishing to bebound by theory, it is believed that, in an embodiment, the performanceof endogenous, non-modified immune effector cells, e.g., T cells or NKcells, is improved. While not wishing to be bound by theory, it isbelieved that, in an embodiment, the performance of a target antigenCAR-expressing cell is improved. In other embodiments, cells, e.g., Tcells or NK cells, which have, or will be engineered to express a CAR,can be treated ex vivo by contact with an amount of an mTOR inhibitorthat increases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or acatalytic inhibitor, is initiated prior to administration of an CARexpressing cell described herein, e.g., T cells or NK cells. In anembodiment, the CAR cells are administered after a sufficient time, orsufficient dosing, of an mTOR inhibitor, such that the level of PD1negative immune effector cells, e.g., T cells or NK cells, or the ratioof PD1 negative immune effector cells, e.g., T cells/PD1 positive immuneeffector cells, e.g., T cells, has been, at least transiently,increased.

In an embodiment, the cell, e.g., T cell or NK cell, to be engineered toexpress a CAR, is harvested after a sufficient time, or after sufficientdosing of the low, immune enhancing, dose of an mTOR inhibitor, suchthat the level of PD1 negative immune effector cells, e.g., T cells, orthe ratio of PD1 negative immune effector cells, e.g., T cells/PD1positive immune effector cells, e.g., T cells, in the subject orharvested from the subject has been, at least transiently, increased.

In one embodiment, the cell expressing a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein, is administered incombination with an agent that ameliorates one or more side effectassociated with administration of a cell expressing a CAR molecule,e.g., an agent described herein.

In one embodiment, the cell expressing a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein, is administered incombination with an agent that treats the disease associated with acancer associated antigen as described herein, e.g., an agent describedherein.

In one embodiment, a cell expressing two or more CAR molecules, e.g., asdescribed herein, is administered to a subject in need thereof to treatcancer. In one embodiment, a population of cells including a CARexpressing cell, e.g., as described herein, is administered to a subjectin need thereof to treat cancer.

In one embodiment, the cell expressing a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein, is administered at a doseand/or dosing schedule described herein.

In one embodiment, the CAR molecule and/or RIAD or Ezrin polypeptide,e.g., as described herein is introduced into immune effector cells(e.g., T cells, NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of cellscomprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein, and one or more subsequent administrations of cellscomprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein, wherein the one or more subsequent administrations areadministered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or 2 days after the previous administration. In one embodiment,more than one administration of cells comprising a CAR molecule and/orRIAD or Ezrin polypeptide, e.g., as described herein are administered tothe subject (e.g., human) per week, e.g., 2, 3, or 4 administrations ofcells comprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g.,as described herein are administered per week. In one embodiment, thesubject (e.g., human subject) receives more than one administration ofcells comprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g.,as described herein per week (e.g., 2, 3 or 4 administrations per week)(also referred to herein as a cycle), followed by a week of noadministration of cells comprising a CAR molecule and/or RIAD or Ezrinpolypeptide, e.g., as described herein, and then one or more additionaladministration of cells comprising a CAR molecule and/or RIAD or Ezrinpolypeptide, e.g., as described herein (e.g., more than oneadministration of the cells comprising a CAR molecule per week) isadministered to the subject. In another embodiment, the subject (e.g.,human subject) receives more than one cycle of cells comprising a CARmolecule, and the time between each cycle is less than 10, 9, 8, 7, 6,5, 4, or 3 days. In one embodiment, the cells comprising a CAR moleculeand/or RIAD or Ezrin polypeptide, e.g., as described herein areadministered every other day for 3 administrations per week. In oneembodiment, the cells comprising a CAR molecule are administered for atleast two, three, four, five, six, seven, eight or more weeks.

In one embodiment, the cells expressing a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein, are administered as afirst line treatment for the disease, e.g., the cancer, e.g., the cancerdescribed herein. In another embodiment, the cells expressing a and/orRIAD or Ezrin polypeptide, e.g., as described herein, are administeredas a second, third, fourth line treatment for the disease, e.g., thecancer, e.g., the cancer described herein.

In one embodiment, a population of cells described herein isadministered.

In another aspect, the invention pertains to the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use as amedicament.

In another aspect, the invention pertains to a the isolated nucleic acidmolecule encoding a CAR of the invention, the isolated polypeptidemolecule of a CAR of the invention, the vector comprising a CAR of theinvention, and the cell comprising a CAR of the invention for use in thetreatment of a disease expressing a cancer associated antigen asdescribed herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described herein,for use as a medicament in combination with a cytokine, e.g., IL-7,IL-15 and/or IL-21 as described herein. In another aspect, the inventionpertains to a cytokine described herein for use as a medicament incombination with a cell expressing a CAR molecule described herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described hereindescribed herein for use as a medicament in combination with a kinaseinhibitor and/or a checkpoint inhibitor as described herein. In anotheraspect, the invention pertains to a kinase inhibitor and/or a checkpointinhibitor described herein for use as a medicament in combination with acell expressing a CAR molecule described herein.

In another aspect, the invention pertains to a cell expressing a CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described herein,for use in combination with a cytokine, e.g., IL-7, IL-15 and/or IL-21as described herein, in the treatment of a disease expressing a tumorantigen targeted by the CAR. In another aspect, the invention pertainsto a cytokine described herein for use in combination with a cellexpressing a CAR molecule described herein, in the treatment of adisease expressing a tumor antigen targeted by the CAR.

In another aspect, the invention pertains to a cell expressing a CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described herein,for use in combination with a kinase inhibitor and/or a checkpointinhibitor as described herein, in the treatment of a disease expressinga tumor antigen targeted by the CAR. In another aspect, the inventionpertains to a kinase inhibitor and/or a checkpoint inhibitor describedherein for use in combination with a cell expressing a CAR moleculedescribed herein, in the treatment of a disease expressing a tumorantigen targeted by the CAR.

In another aspect, the present invention provides a method comprisingadministering a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein, or a cell comprising a nucleic acid encoding a CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described herein. Inone embodiment, the subject has a disorder described herein, e.g., thesubject has cancer, e.g., the subject has a cancer and hastumor-supporting cells which express a tumor-supporting antigendescribed herein. In one embodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating asubject having a disease associated with expression of atumor-supporting antigen as described herein comprising administering tothe subject an effective amount of a cell comprising a CAR moleculeand/or RIAD or Ezrin polypeptide, e.g., as described herein.

In yet another aspect, the invention features a method of treating asubject having a disease associated with expression of atumor-supporting antigen, comprising administering to the subject aneffective amount of a cell, e.g., an immune effector cell (e.g., apopulation of immune effector cells) comprising a CAR molecule and/orRIAD or Ezrin polypeptide, e.g., as described herein, wherein the CARmolecule comprises an antigen binding domain, a transmembrane domain,and an intracellular domain, said intracellular domain comprises acostimulatory domain and/or a primary signaling domain, wherein saidantigen binding domain binds to the tumor-supporting antigen associatedwith the disease, e.g. a tumor-supporting antigen as described herein.

In a related aspect, the invention features a method of treating asubject having a disease associated with expression of atumor-supporting antigen. The method comprises administering to thesubject an effective amount of a cell, e.g., an immune effector cell(e.g., a population of immune effector cells) comprising a CAR moleculeand/or RIAD or Ezrin polypeptide, e.g., as described herein, incombination with an agent that increases the efficacy of the immunecell, wherein:

the CAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain comprising a costimulatory domainand/or a primary signaling domain, wherein said antigen binding domainbinds to the tumor-supporting antigen associated with the disease, e.g.a tumor-supporting antigen as disclosed herein; and the agent thatincreases the efficacy of the immune cell is chosen from one or more of:

a protein phosphatase inhibitor;

a kinase inhibitor;

a cytokine;

an inhibitor of an immune inhibitory molecule; or

an agent that decreases the level or activity of a T_(REG) cell.

In a related aspect, the invention features a method of treating asubject having a disease associated with expression of atumor-supporting antigen, comprising administering to the subject aneffective amount of a cell, e.g., an immune effector cell (e.g., apopulation of immune effector cells) comprising a CAR molecule and/orRIAD or Ezrin polypeptide, e.g., as described herein, wherein:

the CAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain comprising a costimulatory domainand/or a primary signaling domain, wherein said antigen binding domainbinds to the tumor-supporting antigen associated with the disease, e.g.,a tumor-supporting antigen as disclosed herein; and

the antigen binding domain of the CAR molecule has a binding affinity atleast 5-fold less than an antibody from which the antigen binding domainis derived.

In another aspect, the invention features a composition comprising animmune effector cell (e.g., a population of immune effector cells)comprising a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein, for use in the treatment of a subject having a diseaseassociated with expression of a tumor-supporting antigen, e.g., adisorder as described herein.

In any of the aforesaid methods or uses, the disease associated withexpression of the tumor-supporting antigen is selected from the groupconsisting of a proliferative disease, a precancerous condition, acancer, and a non-cancer related indication associated with expressionof the tumor-supporting antigen. In an embodiment, the diseaseassociated with a tumor-supporting antigen described herein is a solidtumor.

In one embodiment of the methods or uses described herein, the CARmolecule and/or RIAD or Ezrin polypeptide, e.g., as described herein, isadministered in combination with another agent. In one embodiment, theagent can be a kinase inhibitor, e.g., a CDK4/6 inhibitor, a BTKinhibitor, an mTOR inhibitor, a MNK inhibitor, or a dual PI3K/mTORinhibitor, and combinations thereof. In one embodiment, the kinaseinhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein,e.g., a CD4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor.The dual PI3K/mTOR inhibitor can be, e.g., PF-04695102.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a CDK4 inhibitor selected from aloisine A; flavopiridol orHMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and thepalbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time,e.g., daily for 14-21 days of a 28 day cycle, or daily for 7-12 days ofa 21 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more cycles of palbociclib are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765);GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059;CNX-774; and LFM-A13. In one embodiment, the BTK inhibitor does notreduce or inhibit the kinase activity of interleukin-2-inducible kinase(ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764;HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and theibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time,e.g., daily for 21 day cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofibrutinib are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a BTK inhibitor that does not inhibit the kinase activityof ITK, e.g., RN-486, and RN-486 is administered at a dose of about 100mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg (e.g., 150 mg, 200 mgor 250 mg) daily for a period of time, e.g., daily a 28 day cycle. Inone embodiment, 1, 2, 3, 4, 5, 6, 7, or more cycles of RN-486 areadministered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus(1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 112), inner salt (SF1126); and XL765.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin isadministered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for 21day cycle, or daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. Inone embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,everolimus and the everolimus is administered at a dose of about 2 mg,2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg,13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g.,daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more cycles of everolimus are administered.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is an MNK inhibitor selected from CGP052088;4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d] pyrimidine (CGP57380);cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine.

In one embodiment of the methods or uses described herein, the kinaseinhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTORinhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF-04691502);N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N′-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

In one embodiment of the methods or uses described herein, a CARexpressing immune effector cell described herein is administered to asubject in combination with a protein tyrosine phosphatase inhibitor,e.g., a protein tyrosine phosphatase inhibitor described herein. In oneembodiment, the protein tyrosine phosphatase inhibitor is an SHP-1inhibitor, e.g., an SHP-1 inhibitor described herein, such as, e.g.,sodium stibogluconate. In one embodiment, the protein tyrosinephosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment of the methods or uses described herein, the CARmolecule is administered in combination with another agent, and theagent is a cytokine. The cytokine can be, e.g., IL-7, IL-15, IL-21, or acombination thereof. In another embodiment, the CAR molecule isadministered in combination with a checkpoint inhibitor, e.g., acheckpoint inhibitor described herein. For example, in one embodiment,the check point inhibitor inhibits an inhibitory molecule selected fromPD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

Methods of Making CAR-Expressing Cells

In another aspect, the invention pertains to a method of making a cell(e.g., an immune effector cell or population thereof) comprisingintroducing into (e.g., transducing) a cell, e.g., a T cell or a NK celldescribed herein, with a vector of comprising a nucleic acid encoding aCAR, e.g., a CAR and/or RIAD or Ezrin polypeptide, e.g., as describedherein; or a nucleic acid encoding a CAR molecule and/or RIAD or Ezrinpolypeptide, e.g., as described herein.

The cell in the methods is an immune effector cell (e.g., a T cell or aNK cell, or a combination thereof). In some embodiments, the cell in themethods is diacylglycerol kinase (DGK) and/or Ikaros deficient.

In some embodiment, the introducing the nucleic acid molecule encoding aCAR and/or RIAD or Ezrin polypeptide, e.g., as described herein,comprises transducing a vector comprising the nucleic acid moleculeencoding a CAR and/or RIAD polypeptide, e.g., as described herein, ortransfecting the nucleic acid molecule encoding a CAR and/or RIAD orEzrin polypeptide, e.g., as described herein, wherein the nucleic acidmolecule is an in vitro transcribed RNA.

In some embodiments, the method further comprises:

providing a population of immune effector cells (e.g., T cells or NKcells); and

removing T regulatory cells from the population, thereby providing apopulation of T regulatory-depleted cells;

wherein steps a) and b) are performed prior to introducing the nucleicacid encoding the CAR to the population. The nucleic acid encoding theCARs can further comprises a RIAD polypeptide or Ezrin polypeptide,e.g., a RIAD polypeptide or Ezrin polypeptide as described herein.

In embodiments of the methods, the T regulatory cells comprise CD25+ Tcells, and are removed from the cell population using an anti-CD25antibody, or fragment thereof. The anti-CD25 antibody, or fragmentthereof, can be conjugated to a substrate, e.g., a bead.

In other embodiments, the population of T regulatory-depleted cellsprovided from step (b) contains less than 30%, 25%, 20%, 15%, 10%, 5%,4%, 3%, 2%, 1% of CD25+ cells.

In yet other embodiments, the method further comprises:

removing cells from the population which express a tumor antigen thatdoes not comprise CD25 to provide a population of T regulatory-depletedand tumor antigen depleted cells prior to introducing the nucleic acidencoding a CAR to the population. The tumor antigen can be selected fromCD19, CD30, CD38, CD123, CD20, CD14 or CD11b, or a combination thereof.

In other embodiments, the method further comprises

removing cells from the population which express a checkpoint inhibitor,to provide a population of T regulatory-depleted and inhibitory moleculedepleted cells prior to introducing the nucleic acid encoding a CAR tothe population. The checkpoint inhibitor can be chosen from PD-1, LAG-3,TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3, and/orCEACAM-5), TIGIT, CTLA-4, BTLA, and LAIR1. Further embodiments disclosedherein encompass providing a population of immune effector cells. Thepopulation of immune effector cells provided can be selected based uponthe expression of one or more of CD3, CD28, CD4, CD8, CD45RA, and/orCD45RO. In certain embodiments, the population of immune effector cellsprovided are CD3+ and/or CD28+.

In certain embodiments of the method, the method further comprisesexpanding the population of cells after the nucleic acid moleculeencoding a CAR has been introduced.

In embodiments, the population of cells is expanded for a period of 8days or less.

In certain embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded in culture for5 days show at least a one, two, three or four fold increase in celldoublings upon antigen stimulation as compared to the same cellsexpanded in culture for 9 days under the same culture conditions.

In yet other embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells exhibit higher proinflammatory IFN-γand/or GM-CSF levels, as compared to the same cells expanded in culturefor 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded by culturingthe cells in the presence of an agent that stimulates a CD3/TCR complexassociated signal and/or a ligand that stimulates a costimulatorymolecule on the surface of the cells. The agent can be a bead conjugatedwith anti-CD3 antibody, or a fragment thereof, and/or anti-CD28antibody, or a fragment thereof.

In other embodiments, the population of cells is expanded in anappropriate media that includes one or more interleukin that result inat least a 200-fold, 250-fold, 300-fold, or 350-fold increase in cellsover a 14 day expansion period, as measured by flow cytometry.

In other embodiments, the population of cells is expanded in thepresence IL-15 and/or IL-7.

In certain embodiments, the method further includes cryopreserving thepopulation of the cells after the appropriate expansion period.

In yet other embodiments, the method of making disclosed herein furthercomprises contacting the population of immune effector cells with anucleic acid encoding a telomerase subunit, e.g., hTERT. The nucleicacid encoding the telomerase subunit can be DNA.

The present invention also provides a method of generating a populationof RNA-engineered cells, e.g., cells described herein, e.g., immuneeffector cells (e.g., T cells, NK cells), transiently expressingexogenous RNA. The method comprises introducing an in vitro transcribedRNA or synthetic RNA into a cell, where the RNA comprises a nucleic acidencoding a CAR molecule and/or RIAD or Ezrin polypeptide, e.g., asdescribed herein.

In another aspect, the invention pertains to a method of providing ananti-tumor immunity in a subject comprising administering to the subjectan effective amount of a cell comprising a CAR molecule and/or RIAD orEzrin polypeptide, e.g., as described herein. In one embodiment, thecell is an autologous T cell or NK cell. In one embodiment, the cell isan allogeneic T cell or NK cell. In one embodiment, the subject is ahuman

In one aspect, the invention includes a population of autologous cellsthat are transfected or transduced with a vector comprising a nucleicacid molecule encoding a CAR molecule, e.g., as described herein. In oneembodiment, the vector is a retroviral vector. In one embodiment, thevector is a self-inactivating lentiviral vector as described elsewhereherein.

In one embodiment, the vector is delivered (e.g., by transfecting orelectroporating) to a cell, e.g., a T cell or a NK cell, wherein thevector comprises a nucleic acid molecule encoding a CAR and/or RIAD orEzrin polypeptide, e.g., as described herein, which is transcribed as anmRNA molecule, and the CARs of the present invention is translated fromthe RNA molecule and expressed on the surface of the cell.

In another aspect, the present invention provides a population ofCAR-expressing cells and/or RIAD or Ezrin polypeptide expressing cells,e.g., as described herein, e.g., CAR-expressing immune effector cells(e.g., T cells or NK cells). In some embodiments, the population ofCAR-expressing cells comprises a mixture of cells expressing differentCARs. For example, in one embodiment, the population of CAR-expressingimmune effector cells (e.g., T cells or NK cells) can include a firstcell expressing a CAR having an antigen binding domain that binds to afirst tumor antigen as described herein, and a second cell expressing aCAR having a different antigen binding domain that binds to a secondtumor antigen as described herein. As another example, the population ofCAR-expressing cells can include a first cell expressing a CAR thatincludes an antigen binding domain that binds to a tumor antigen asdescribed herein, and a second cell expressing a CAR that includes anantigen binding domain to a target other than a tumor antigen asdescribed herein. In one embodiment, the population of CAR-expressingcells includes, e.g., a first cell expressing a CAR that includes aprimary intracellular signaling domain, and a second cell expressing aCAR that includes a secondary signaling domain, e.g., a costimulatorysignaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having anantigen binding domain that binds to a tumor antigen as describedherein, and a second cell expressing another agent, e.g., an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4,TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, theagent which inhibits an inhibitory molecule, e.g., is a moleculedescribed herein, e.g., an agent that comprises a first polypeptide,e.g., an inhibitory molecule, associated with a second polypeptide thatprovides a positive signal to the cell, e.g., an intracellular signalingdomain described herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD-1, LAG-3,CTLA-4, CD160, BTLA, LAIR1, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3and/or CEACAM-5), 2B4 and TIGIT, or a fragment of any of these, and asecond polypeptide which is an intracellular signaling domain describedherein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27 orCD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD-1 or a fragment thereof,and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28, CD27, OX40 or 4-IBB signaling domain describedherein and/or a CD3 zeta signaling domain described herein).

In one embodiment, the nucleic acid molecule encoding a CAR of thepresent invention molecule, e.g., as described herein, is expressed asan mRNA molecule. In one embodiment, the genetically modified CAR of thepresent invention-expressing cells, e.g., immune effector cells (e.g., Tcells, NK cells), can be generated by transfecting or electroporating anRNA molecule encoding the desired CARs (e.g., without a vector sequence)into the cell. In one embodiment, a CAR of the present inventionmolecule is translated from the RNA molecule once it is incorporated andexpressed on the surface of the recombinant cell.

In another aspect, the invention features a composition including anucleic acid molecule encoding any of the foregoing CARs and a nucleicacid molecule encoding an RIAD or Ezrin polypeptide.

In certain aspects, the RIAD polypeptide includes, e.g., a polypeptidehaving an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 63. In related aspects, the RIADpolypeptide can be any of the “anchoring disruption molecule or AKAPmimic” disclosed in U.S. Patent Application Publication No. 20080248008,each of which is hereby specifically incorporated by reference. Forexample (as described the '008 publication), the RIAD polypeptide canhave the following sequence: L E Q Y A N Q L A D Q I I K E A T E, (SEQID NO: 63), i.e., X1=L, X2=E, X3=Q, X4=N, X5=Q, X6=D, X7=Q, X8=I, X9=K,X10=E, X11=A, X12=T and X13=E, or a variant of this sequence wherein thevariant has a substitution at any one, two, three, four or five ofpositions X1 to X13 of the sequence. Preferably the one or moresubstitutions are selected from X1=F, Y, I, V, W or C; X2=C, D, R or K;X3=F, K, R, A, I, L, M, S, T, V, G, N, W, D or E; X4=K, R, G, T, S, W, Dor E; X5=S, F, K, R, M, W, Y, D or E; X6=E, I, A, R, S, F, H, W, K, L,Y, M, N, Q or G; X7=K, R, F, N, S, T, V, L, M, W, I, D or E; X8=V; X9=D,E, L, S, R, A, M, T, W, Y; X10=D, R, K or Q; X11=I or V; X12=F, C, M, K,R, I or L; and X13=D, N, V, Y, G, H, I, Q, A, F, K, L, M, R, S, T or W.

X5 can also be L or T and X11 can also be C.

Alternatively, the one or more substitutions are selected from X1=C, Ior F; X2=D, K, R, V, A, I, L, Q, S or T; X3=D, E, A, I, S or V; X4=D, E,S, A, M or Q; X5=D, E, C, I, M or F; X6=G, S, E or M; X7=D, E, I or M;X8=V, D or E; X9=A, D, E, M, R, T, W or Y; X10=D; X12=L, V or W; andX13=D, R, K or W.

Especially preferably,

(i) single amino acid substitutions are selected from the groupconsisting of X1=C; X2=D; X3=D or E; X4=D or E; X5=D or E; X6=E, G or S;X7=D, E or I; X9=D, E, A, M, R, T, W or Y; X10=D; and X13=D (referred toas L1C, E2D, Q3D, Q3E, N6D, N6E, Q7D, Q7E, D10E, D10G, MOS, Q11D, Q11E,Q11I, K14D, K14E, K14A, K14M, K14R, K14T, K14W, K14Y, E15D and E18Drespectively); further single amino acid substitutions are selected fromX1=F, I or Y; X3=A, K, M, R, S or T; X4=G or T; X5=K, L M, R, S or T;X6=N; X7=K, L or R; X12=C or M; X13=M, N, Q or T (L1F, L1I, L1Y, Q3A,Q3K, Q3M, Q3R, Q11K, Q11L, Q11R, T17C, T17M, Q3S, Q3T, N6G, N6T, Q7K,Q7L, Q7M, Q7R, Q7S, Q7T, D10N, E18M, E18N, E18Q, E18T); and

(ii) double amino acid substitutions of said sequence are selected fromthe group consisting of:

a) when X6 is S, either X2 is K or D, X5 is D or E or X8 is V(alternatively referred to as D10S E2K, D10S E2D, D10S Q7D, D10S Q7E orD10S I12V);

b) when X6 is E, either X2 is K or D, X5 is D or E or X8 is V(alternatively referred to as D10E E2K, D10E E2D, D10E Q7D, D10E Q7E,D10E I12V); and

c) when X8 is V, either X2 is K or D, X6 is M or X12 is L (referred toas I12V E2K, I12V E2D, I12V D10M or I12V T17L);

alternatively, double amino acid substitutions of said sequence may beselected from the group consisting of:

a) when X3 is A, either X4 is G; X5 is K, M or R; X7 is K, L or R; orX13 is M, N or Q;

b) when X13 is T, either X1 is F; X3 is A, K, E, M, R or S; X4 is T orG; X5 is M or R; X7 is R or K; or X9 is R;

c) when X13 is Q, either X3 is A, S, E, K, M, R or S; X4 is G; X5 is M,K or R; or X7 is K; or

d) when X13 is M, either X3 is E, M, R, S or K; X4 is G; X5 is M, R orK; X7 is R; or X12 is C or M;

e) when X7 is K, either X3 is A, R, E, K or M; X4 is G; X5 is M; X9 isR; X12 is C or M; or X13 is Q, T, M or N; and

f) when X5 is M, either X7 is K; or X13 is N, Q or T;

(iii) triple amino acid substitutions of said sequence are selected fromthe group consisting of:

a) when X2 is K, X4 is E and X6 is S (E2K N6E D10S);

b) when X2 is D, X4 is D or E and X6 is E or S (E2D N6D/N6E D10E/D10S);

c) when X2 is K, X6 is M and X8 is V (E2K D10M I12V);

d) when X2 is D, X6 is E or M and X8 is D, E or V (E2D D10E/D10MI12D/I12E/I12V);

e) when X7 is D, X8 is V and X12 is L (Q11D I12V T17L);

f) when X7 is E, X8 is V and X12 is L (Q11E I12V T17L);

g) when X6 is S, X8 is V and either

X1 is I or F;

X2 is A, I, L, Q, S, T, D or V;

X3 is E, D, S, A, I or V;

X4 is S, E, D, A, M or Q;

X5 is M, D, E, I or F;

X7 is D, E, M;

X9 is R;

X10 is D;

X12 is W or L; or

X13 is K or W; and

h) when X6 is E, X8 is V and either

X1 is I or F;

X2 is A, I, L, Q, S, T, D or V;

X3 is E, D, S, A, I or V;

X4 is S, E, D, A, M or Q;

X5 is M, D, E, I or F;

X7 is D, E, M;

X9 is R;

X10 is D;

X12 is W or L; or

X13 is K or W; and

iv) quadruple amino acid substitutions of said sequence are selectedfrom the group consisting of:

a) when X5 is E, X6 is S, X8 is V and either

X3 is A, D, E or S;

X4 is E, D or S;

X12 is L or W;

b) when X5 is D, X6 is S, X8 is V and either

X3 is A, D, E or S;

X4 is E, D or S;

X12 is L or W;

c) when X4 is E, X6 is S or E, X8 is V and X2 is either K, D, V, A, I,L, Q, S or T;

d) when X4 is D, X6 is S or E, X8 is V and X2 is either K, D, V, A, I,L, Q, S or T;

e) when X2 is K, X6 is S, X8 is V and either

X1 is F;

X3 is E, D, A or S;

X4 is S, A, M, D or E;

X5 is F, I, M, D or E;

X7 is M, D or E;

X12 is L or W; or

X13 is D or K;

f) when X2 is D, X6 is S, X8 is V and either

X1 is F;

X3 is E, D, A or S;

X4 is S, A, M, D or E;

X5 is F, I, M, D or E;

X7 is M, D or E;

X12 is L or W; or

X13 is D or K;

g) when X2 is V; X3 is E or D, X6 is S or E and X8 is V;

h) when X2 is D; X3 is E or D, X6 is S or E and X8 is V;

i) when X2 is K; X4 is E or D, X6 is M or E and X8 is V; and

j) when X2 is D; X4 is E or D, X6 is M or E and X8 is V; and

v) quintuple amino acid substitutions of said sequence are selected fromthe groups consisting of:

a) when X2 is K, X4 is E or D, X6 is S or E, X8 is V and either

X1 is I or F;

X3 is D, E, A, I, S or V;

X5 is M, F, D, E or I;

X7 is D, E or M;

X9 is R;

X11 is D;

X12 is L or W; or

X13 is K, D or W;

b) when X2 is D, X4 is E or D, X6 is S or E, X8 is V and either

X1 is I or F;

X3 is D, E, A, I, S or V;

X5 is M, F, D, E or I;

X7 is D, E or M;

X9 is R;

X10 is D;

X12 is L or W; or

X13 is K, D or W;

c) when X2 is A, X4 is E or D, X5 is C, D or E, X6 is S or E and X8 isV;

d) when X2 is D, X4 is E or D, X5 is C, D or E, X6 is S or E and X8 isV;

e) when X2 is T, X4 is E or D, X5 is E or D, X6 is S or E and X8 is V;

f) when X2 is Q, X4 is E or D, X5 is E or D, X6 is S or E and X8 is V;and

or a peptidomimetic or analogue thereof.

Particularly preferred RIAD polypeptides are polypeptides comprising theabove sequence or the sequence with a single amino acid substitutionparticularly as described above.

Highly preferred are polypeptides comprising the above sequence with asingle amino acid substitution selected from the group consisting ofX7=D or E or X9=A, M, T, W or Y.

In a further preferred feature, the RIAD polypeptide of the invention isa polypeptide of SEQ ID NO: 72 in which

X1=L, X2=K, X3=Q, X4=N, X5=Q, X6=S, X7=Q, X8=V, X9=K, X10=E, X11=A,X12=T and X13=E,

i.e. comprising the following sequence:

(SEQ ID NO: 72) L K Q Y A N Q L A S Q V I K E A T E

or a variant thereof, wherein said variant has a substitution at anyone, two, three or four of positions X1 to X13 of said sequence.

Preferably said one or more substitutions are selected from X1=F or I;X2=A, E, D, R, V, Q, S, I, L or T; X3=A, S, E, D, V or I; X4=A, M, S, E,D or Q, X5=F, I, M, D or E; X6=M, E or D; X7=M, E or D; X8=I; X9=R or C;X10=D; X11=C; X12=L, C or W; and X13=D or the substitutions defined forthe previous preferred sequence.

Alternatively, said one or more substitutions are selected from X1=C, Ior F; X2=D, R, V, A, I, L, Q, S, E or T; X3=D, E, A, I, S or V; X4=D, E,S, A, M or Q; X5=D, E, I, M or F; X6=D, E, G or M; X7=D, E, I or M;X8=I; X9=A, D, E, M, R, T, W or Y; X10=D; X12=L or W; and X13=K, R, D orW.

Especially preferably

(i) single amino acid substitutions of the sequence are selected fromthe group consisting of X2=A, D, E, V, Q, S, I, L or T; X3=D, E, S or A;X4=D, E, A, S or M; X5=D, E or M; X6=D or E; X7=D or E; X8=I; X9=C;X10=D; X12=W or L; and X13=D; and

(ii) double amino acid substitutions of said sequence are selected fromthe group consisting of:

(a) when X2 is E or D, either

X1 is I or F;

X3 is A, E, D, S, I or V;

X4 is A, E, D, S, M or Q;

X5 is F, I, D, E or M;

X6 is M, D or E;

X7 is D, E or M;

X8 is I;

X9 is R;

X10 is D;

X12 is L or W; or

X13 is D, K or W;

(b) when X6 is E or D, either

X1 is I or F;

X2 is A, D, E, S, I, V, L, Q or T;

X3 is A, D, E, S, I or V;

X4 is D or E;

X5 is F, I, D, E or M;

X6 is M, D or E;

X7 is M, D or E;

X8 is I;

X9 is R;

X10 is D;

X12 is L or W; or

X13 is W, K or D; and

(c) when X2 is V, X3 is E or D; and

(iii) triple amino acid substitutions of said sequence are selected fromthe group consisting of:

(a) when X2 is E or D and X5 is E or D, either

X3 is S, D, E or A;

X4 is E, D or S;

X8 is I; or

X12 is W or L;

(b) X2 is T and X4 and X5 are both E or D;

(c) X2 is A, X4 is E or D and X5 is E or D;

(d) X2 is E or D, X6 is D or E and either X8 is I or X12 is L;

(e) X2 is E or D, X6 is G and X8 is I; (f) X2 is V, X4 is E or D and X5is E or D; (g) X2 is D or E, X6 is D or E and X8 is I; (h) X2 is Q, X4is E or D and X5 is E or D; and (i) X2 is E, X6 is N and X8 is I; and

(iv) quadruple amino acid substitutions of said sequence are selectedfrom the group consisting of:

(a) when X2 is E or D, X6 is D or E and X, is I, either

X1 is C;

X3 is D or E;

X4 is D or E;

X5 is D or E;

X7 is D, E or I; or

X9 is A, D, E, M, R, T, W or Y;

X12 can be C or M;

X13 can be M, N, Q or T; and

X1 can also be F, I or Y;

X3 can also be A, K, M, R, S or T;

X4 can also be G or T;

X5 can also be K, L, M, R, S or T;

X7 can also be K, L or R;

(b) X2 is E or D, X6 and X7 are both D or E and X12 is L, or apeptidomimetic or analogue thereof.

Alternatively, the polypeptide may consist of any of the abovesequences, or a 1-6, e.g. 1, 2, 3 or 4 (preferably 1 or 2) amino acid N-or C-terminal (preferably C-terminal) truncation of said polypeptide,provided that said truncated polypeptide retains the ability to bind toPKA I. Such polypeptides fall within the scope of derivatives asdescribed herein. Examples of truncated polypeptides which areparticularly useful are:

(SEQ ID NO: 80) LVQYAEQLASQVIKEAT (SEQ ID NO: 81) LESYANQLASQVIKEAT (SEQID NO: 82) LESYASQLASQVIKEAT (SEQ ID NO: 83) LEQYAEQLASQVIKEAT (SEQ IDNO: 84) LEQYAEQLASQVIKEA (SEQ ID NO: 85) LVQYAEELASQVIKEAT (SEQ ID NO:86) LVQYAEELASQVIKEA (SEQ ID NO: 87) LESYANELASQVIKEAT (SEQ ID NO: 88)LESYANELASQVIKEA (SEQ ID NO: 89) LEQYASELASQVIKEAT (SEQ ID NO: 90)LEQYAEELASQVIKEAT (SEQ ID NO: 91) LEQYAEELASQVIKEA (SEQ ID NO: 92)LEQYANELASQIIKEAT (SEQ ID NO: 93) LEQYANELASQIIKEA (SEQ ID NO: 94)LEQYANELASQVIKEAL (SEQ ID NO: 95) LEQYANELASQVIKEA (SEQ ID NO: 96)LEQYANQLASQIIKEAT (SEQ ID NO: 97) LEQYANQLASQIIKEA (SEQ ID NO: 98)LEQYANQLASQVIKEAL (SEQ ID NO: 99) LEQYANQLASQVIKEA (SEQ ID NO: 100)LEQYANQLADQIIKEAT (SEQ ID NO: 101) LEQYANQLADQIIKEA (SEQ ID NO: 102)LEQYANQLADDVIKEAL (SEQ ID NO: 103) LEQYANQLADDVIKEA (SEQ ID NO: 104)LEQYANQLADDIIKEAT (SEQ ID NO: 105) LEQYANQLADDIIKEA (SEQ ID NO: 106)LEQYANQLADDVIKEAL (SEQ ID NO: 104) LEQYANQLADDVIKEA

In certain embodiments, the RIAD polypeptide also includes a RISRsubunit, e.g., a polypeptide having an amino acid sequence having atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:64 (e.g., a polypeptide having 100% identity to SEQ ID NO: 64). Inpreferred embodiments, the inclusion of a RISR subunit increases theaffinity and/or selectivity of the RIAD polypeptide for PKA type I(including, e.g., the molecules described in Jarnaess et al., J. Biol.Chem. 283:33708 (2008) which are each incorporated by reference).

In certain aspects, the foregoing chimeric antigen receptor and RIADpolypeptides are encoded by a single nucleic molecule in the same frameand as a single polypeptide chain (e.g., the RIAD polypeptide isattached to the N-terminus or C-terminus of the chimeric antigenreceptor). In this aspect, the RIAD polypeptide and chimeric antigenreceptor can, e.g., be separated by one or more peptide cleavage sites.(e.g., an auto-cleavage site or a substrate for an intracellularprotease). Examples of peptide cleavage sites include the following,wherein the GSG residues are optional:

T2A: (SEQ ID NO: 73) (GSG) E G R G S L L T C G D V E E N P G P P2A: (SEQID NO: 74) (GSG) A T N F S L L K Q A G D V E E N P G P E2A: (SEQ ID NO:75) (GSG) Q C T N Y A L L K L A G D V E S N P G P F2A: (SEQ ID NO: 76)(GSG) V K Q T L N F D L L K L A G D V E S N P G P

In a related aspect, the invention features a single protein, asdescribed above, encoding a CAR and RIAD or Ezrin polypeptide.

In other aspects, the foregoing chimeric antigen receptor and RIAD orEzrin polypeptides are encoded by a single, or multiple, nucleicmolecules and are not expressed as a single polypeptide. Here, e.g., thechimeric antigen receptor and the RIAD or Ezrin polypeptide can becontrolled by a common promoter or be separated by an internal ribosomalentry site. Alternatively, the expression of the chimeric antigenreceptor and the RIAD polypeptide can be, e.g., controlled by separatepromoters.

In yet another aspect, the invention features one or more vectors (e.g.,any of the vectors described above) including the foregoing nucleic acidmolecules encoding a CAR and RIAD or Ezrin polypeptide. As noted above,the vector can also include a promoter chosen from an EF-1 promoter(e.g., including a sequence of SEQ ID NO: 1), a CMV IE gene promoter, anEF-1α promoter, an ubiquitin C promoter, or a phosphoglycerate kinase(PGK) promoter.

In yet another aspect, the invention feature an immune effector cell (ahuman T cell (e.g., CD8+ T cell) or a human NK cell, optionally, whereinthe T cell is diacylglycerol kinase (DGK) and/or Ikaros deficient)),including

-   -   (i) the nucleic acid molecule encoding a chimeric antigen        receptor and nucleic acid molecule encoding an RIAD or Ezrin        polypeptide, as described above;    -   (ii) a foregoing polypeptide including a CAR and a RIAD or Ezrin        polypeptide; and/or    -   (iii) a vector encoding the above constructs.

The invention also features a method of making a chimeric antigenreceptor and RIAD or Ezrin polypeptide-expressing immune effector cell(e.g., a population of chimeric antigen receptor-expressing immuneeffector cells), including introducing any of the foregoing nucleicacids and/or vectors, into an immune effector cell, under conditionssuch that the chimeric antigen receptor molecule is expressed.

As noted above, this method can, optionally, further include

-   -   a) providing a population of immune effector cells (e.g., T        cells or NK cells); and    -   b) removing T regulatory cells from the population, thereby        providing a population of T regulatory-depleted cells;

wherein steps a) and b) are performed prior to introducing the nucleicacid (or nucleic acids) encoding the chimeric antigen receptor and RIADor Ezrin polypeptide to the population.

In yet another embodiment, the invention features any and all of theforegoing methods of providing anti-tumor immunity in a subject, whereinthose methods also feature administering to the subject an effectiveamount of the foregoing immune effector cells.

The invention also features, in the place of, or in addition to, any ofthe foregoing RIAD polypeptides, a peptide comprising an amphipathichelix domain and at least one cluster of basic amino acids, wherein thepeptide disrupts protein kinase A (PKA) and A-kinase anchoring protein(AKAP) association, e.g., wherein the amphipathic helix domain and thecluster of basic amino acids bind the PKA. Such a peptide can be, e.g.,a fragment of an AKAP, wherein the fragment comprises an amphipathichelix domain of the AKAP. The term “A-kinase anchoring protein” or“AKAP” should be construed to include any one of AKAP1, AKAP2, AKAP3,AKAP4, AKAP5, AKAP6, AKAP7, AKAP8, AKAP9, AKAP10, AKAP11, AKAP12,AKAP13, AKAP15, AKAP18, AKAP28, AKAP75, AKAP78, AKAP79, AKAP80, AKAP82,AKAP84, AKAP95, AKAP110, AKAP121, AKAP140, AKAP149, AKAP150, AKAP220,AKAP350, AKAP450, AKAP-KL, AKAP-Lbc, DAKAP-1, mAKAP, T-AKAP80, BIG2,Ezrin, CG-NAP, Gravin, Ht31, Hyperion, MAP2B, MAP2D, Merlin, myeloidtranslocation gene 8, myeloid translocation gene 16b, Myospryn, MyosinVIIA, MyRIP, Neurobeachin, PAP7, Pericentrin, Rab32, Rt31, SFRS17A,SKIP, SSeCKS, Synemin, WAVE-1, and Yotiao. In such aspects, the peptidecan have a length of, e.g., about 10 amino acids to about 60 amino acidsin length.

By “amphipathic helix domain” refers to an A-kinase binding (AKB) domainof an AKAP that binds the dimerization and docking (D/D) domain of PKAthat localizes to a particular location within the cell, such as thecell membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the “regulatory subunit I specifierregion/regulatory subunit I anchoring disruptor” (RISR-RIAD) sequenceconstruct that was cloned into the mesoCAR plasmid, separated by a T2Asequence to yield stoichiometric expression of both CAR and RISR-RIAD.

FIG. 2 is a blot showing that RISR-RIAD expression prevented proteinkinase A (PKA) from inhibiting Lck and thus prevented immunosuppression.

FIG. 3 is a blot showing that RISR-RIAD expression resulted in moreactive signaling at baseline and after TCR stimulation.

FIG. 4 is a graph showing killing of antigen-expressing cells (AE17meso)and control cells (AE17ova) that were incubated overnight in thepresence of varying effector-to-target ratios (E:T); e.g., 10CAR-expressing T cells to 1 tumor cell. Cytolytic ability of these Tcells was then quantified.

FIG. 5A is a graph showing suppression of killing of AE17meso cells thatwere incubated overnight with both CAR T cells at the indicated E:T inthe presence of varying concentrations of PGE2. A dose-dependentsuppression of killing by mesoCAR T cells was observed, but not bymesoCAR-RISR-RIAD T cells.

FIG. 5B is a graph showing suppression of killing of AE17meso cells thatwere incubated overnight with both CAR T cells at the indicated E:T inthe presence of varying concentrations of adenosine. A dose-dependentsuppression of killing by mesoCAR T cells was observed, but not bymesoCAR-RISR-RIAD T cells.

FIG. 6 is a series of schematic representations of viral-engineeredconstructs for transduction of T cells. A) The previously describedmurine mesothelin (SS1)-binding chimeric receptor, along with 4-1BB(CD137) and CD3ζ intracellular signaling domains of human origin, ormesoCAR, and RISR-RIAD were cloned into the retroviral MigR1 vector fortransduction of primary murine T cells. B) Similarly, anti-FAP scFv,intracellular 4-1BB and CD3ζ of murine origin, and RISR-RIAD were clonedinto the MigR1 vector for transduction of primary murine T cells. C) Fortransduction of primary human T cells, mesoCAR and RISR-RIAD (separatedby a T2A sequence for stoichiometric protein expression) were clonedinto the lentiviral pTRPE backbone. D) NY-ESO1 LY95 transgenic TCRconstruct as expressed in the lentiviral pTRPE backbone.

FIG. 7A is a flow plot showing that primary murine T cells transducedwith mesoCAR-RISR-RIAD resisted immunosuppression and killed moreeffectively in vitro. Primary murine T cells isolated and transducedwith mesoCAR-RISR-RIAD; transduction efficiency was determined byprimary staining using a biotinylated goat anti-mouse IgG recognizingthe F(ab′)₂ fragment, followed by a secondary PE-conjugated streptavidinantibody, along with detection of myc-tagged RISR-RIAD.

FIG. 7B is a graph showing various effector-to-target (E:T) ratios ofmesoCAR and mesoCAR-RISR-RIAD T cells co-cultured with eitherova-expressing AE17 (AE17ova) or mesothelin-expressing AE17 (AE17meso)cells overnight. Statistical analysis was performed using two-way ANOVAcomparing the percent cytolysis of AE17meso cells by mesoCAR versusmesoCAR-RISR-RIAD T cells.

FIG. 7C is a graph showing cell culture supernatant from the overnightcytolysis assay measured for IFNγ production via ELISA, and quantifiedto compare production by mesoCAR and mesoCAR-RISR-RIAD T cells.

FIG. 7D is a panel of graphs showing an overnight cytolysis assayagainst AE17meso to assess resistance of mesoCAR and mesoCAR-RISR-RIAD Tcells to increasing doses of adenosine (upper graph) shown at 10:1 E:Tand increasing doses of PGE2 (lower graph) shown at the E:T of 10:1.Statistics were performed using one-way ANOVA comparing the extent ofcytolysis inhibition in the presence of adenosine versus no inhibitor.

FIG. 8A is a panel of graphs showing enhanced therapeutic responses ofmurine mesoCAR-RISR-RIAD T cells. Two million AE17meso cells weresubcutaneously injected into the right flanks of wild-type C57Bl/6 mice.When tumors were approximately 200 mm³ in volume, 10 million mesoCAR-and mesoCAR-RISR-RIAD-expressing T cells were injected via the tailvein, and tumor development was monitored using calipers. At Day 10after T cell injections, animals were sacrificed for ex vivo analysis.

FIG. 8B is a panel of graphs showing enhanced therapeutic responses ofmurine mesoCAR-RISR-RIAD T cells. One million PDA4662 cells weresubcutaneously injected into wild-type mice, and 10 million FAPCAR- andFAPCAR-RISR-RIAD-expressing T cells were injected via the tail vein.Mice were monitored and sacrificed at Day 23 post-T cell administration.

FIG. 9A is a panel of images showing that mesoCAR-RISR-RIAD T cellsexhibited superior tumor infiltration capacity in an integrin-mediatedmanner. AE17meso tumors harvested from mice at Day 10 were digested, andsingle cell suspensions were prepared for flow cytometry analyses. Totaltumor digest were stained for CD4 and CD8.

FIG. 9B is a panel of images showing spleens isolated fromAE17meso-bearing mice, and processed for flow cytometry analyses. Singlecell suspensions were stained for CD4 and CD8 cells.

FIG. 9C is a graph showing equal number of both CAR T cells placed intranswell inserts, and allowed to migrate toward the indicatedstimulants placed in the bottom well. After 4 hours, migrated cells, andcells that remained in the transwells were collected, counted, andstained for different adhesion and migration markers.

FIG. 9D is a graph showing that mesoCAR-RISR-RIAD T cells exhibited muchhigher integrin ß1 (CD29) expression compared to mesoCAR T cells.

FIG. 10 is a graph showing suppression of killing of EM-meso cells thatwere incubated overnight with both CAR T cells at the indicated E:T inthe presence of varying concentrations of adenosine. A dose-dependentsuppression of killing by mesoCAR T cells was observed, but not bymesoCAR-RISR-RIAD T cells.

FIG. 11A is a panel of graphs showing that human T cells transduced withmesoCAR-RISR-RIAD exhibit superior killing ability and robust IFNγproduction in vitro. Lentiviral-transduced primary human T cells asdetected by a primary biotinylated goat anti-mouse IgG recognizing theF(ab′)₂ fragment, followed by a secondary APC-Cy7-conjugatedstreptavidin antibody, along with detection of myc-tagged RISR-RIAD.

FIG. 11B is a graph showing various effector-to-target (E:T) ratios ofmesoCAR and mesoCAR-RISR-RIAD T cells that were co-cultured with eitherparental EM (EMP) or mesothelin-expressing EM (EMmeso) cells overnight.Statistical analysis was performed using two-way ANOVA comparing thepercent cytolysis of EMmeso cells by mesoCAR versus mesoCAR-RISR-RIAD Tcells.

FIG. 11C is a graph showing IFNγ production via ELISA from cell culturesupernatant taken from the overnight cytolysis assay, and quantificationof IFNγ performed by comparing mesoCAR and mesoCAR-RISR-RIAD T cells.

FIG. 12 is panel of graphs showing resistance of mesoCAR-RISR-RIAD Tcells to immunosuppression. The upper graph shows the contribution ofthe RISR-RIAD transgene against immunosuppression of the effector T cellpopulation, mesoCAR and mesoCAR-RISR-RIAD human T cells when co-culturedwith EMmeso cells overnight in the presence of increasing doses ofadenosine; shown is the E:T at 5:1. Statistics were performed usingone-way ANOVA comparing the extent of mesoCAR immunosuppression in thepresence of adenosine versus no inhibitor. The lower graph shows thekilling of EMmeso cells by mesoCAR and mesoCAR-RISR-RIAD T cells whenchallenged with the addition of increasing doses of PGE2; shown is theE:T at 5:1.

FIG. 13A is a panel of images showing signaling in mesoCAR andmesoCAR-RISR-RIAD human T cells. Equal numbers of mesoCAR- andmesoCAR-RISR-RIAD-expressing T cells were exposed to immobilized CD3 andCD28 antibodies for 5 and 20 minutes. Lysates were prepared andimmunoblotted for phospho-ERK (pERK), phospho-Lck at tyrosine-394(pLck-Y394), and phospho-Akt (pAkt), along with their respective loadingcontrols, including actin. Bar charts show densitometry quantificationof the indicated protein.

FIG. 13B is a blot showing lysates of human T cells immunoblotted forphospho-Csk at serine 364 (pCsk-S364), and pLck at tyrosine 505(pLck-Y505) with actin as loading control.

FIG. 14A is a panel of graphs showing tumor control of primary humanmesoCAR and mesoCAR-RISR-RIAD T cells in vivo. Two million EMmeso cellswere injected subcutaneously into the right flanks of immunodeficientNSG mice, and after they were established (around 200 mm³ in volume), 10million mesoCAR- or mesoCAR-RISR-RIAD-expressing T cells wereadministered via tail vein injections. Tumor development was monitoredusing calipers for the next 32 days after T cell administration, andanimals were then sacrificed for ex vivo analyses.

FIG. 14B is a panel of images showing analysis of tumors harvested frommice at Day 32. The tumors were digested, and single cell suspension forflow cytometry analysis was prepared. Cells were stained with anti-CD8and live/dead antibodies to determine the frequency of live CD8 cellswithin the tumors.

FIG. 14C is a panel of graphs showing tumor-infiltrating lymphocytes(TILs) isolated from mesoCAR- and mesoCAR-RISR-RIAD-treated tumorssubjected to an overnight cytolysis assay at a 10:1 E:T ratio againstEMmeso cells. The upper graph shows mesoCAR and mesoCAR-RISR-RIAD Tcells prepared from the same batch that had been frozen away (“cryo”).The cytolysis assay was performed immediately after TIL isolation (“atharvest”), and “after overnight rest” in cell culture medium. The lowergraph shows cell culture supernatant from the overnight cytolysis assayand assayed for IFNγ production via ELISA, and quantification of IFNγwas performed comparing mesoCAR and mesoCAR-RISR-RIAD T cells.

FIG. 15 is a graph showing the ability of the Ly95-RISR-RIAD constructto kill tumor cells was enhanced at each E:T ratio.

FIG. 16 is a graph showing that the Ly95-RISR-RIAD construct was lesssusceptible to inhibition by low dose PGE2 than the Ly95 T cells.

FIG. 17 is a graph showing that the Ly95-RISR-RIAD construct was lesssusceptible to inhibition by high dose PGE2 than the Ly95 T cells

FIG. 18 is a graph showing that the Ly95-RISR-RIAD construct was lesssusceptible to inhibition by adenosine than the Ly95 T cells.

FIG. 19 is a graph showing that Ly95-RISR-RIAD (lower graph) producedmore IFNgamma than Ly95 T cells (upper graph) under adenosinesuppression conditions.

FIG. 20 is a graph showing that Ly95-RISR-RIAD (lower graph) producedmore IFNgamma than Ly95 T cells (upper graph) under low dose PGE2suppression conditions.

FIG. 21 is a graph showing that Ly95-RISR-RIAD (lower graph) producedmore IFNgamma than Ly95 T cells (upper graph) under high dose PGE2suppression conditions.

FIG. 22 is a schematic showing that the RIAD peptide specifically bindsto PKA and displaces them from the lipid membrane, therefore abrogatingPKA-mediated signaling.

FIG. 23 is a graph showing tumor volume as a function of time inAE17meso-bearing mice were treated with mesoCAR and mesoCAR-riad Tcells.

FIG. 24 is a graph showing human interferon expression in cells treatedwith the indicated constructs. ELISA depicting interferon-γ productionfrom mesoCAR and mesoCAR-riad TILs co-cultured with EMM tumor cellsovernight at a E:T of 10:1.

DETAILED DESCRIPTION

In general, the invention features the expression of a RIAD polypeptide(e.g., RIAD, RISR, and/or RISR-RIAD) in a T-cell containing a chimericantigen T cell receptor. This invention is based, at least in part, onthe discovery that the co-expression of a chimeric antigen receptor anda RIAD polypeptide (e.g., RIAD, RISR, and/or RISR-RIAD) in T cellsresult in increased killing of tumor cells both in vitro and in vivo.The invention also features the use of Ezrin-derived polypeptides for asimilar purpose.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “RIAD polypeptide” is defined as a polypeptide comprising a PKAI anchoring disrupting molecule or AKAP mimic which comprises thefollowing amino acid sequence (as set forth in U.S.: (SEQ ID NO: 77):

X1 X2 X3 Y A X4 X5 L A X6 X7 X8 I X9 X10 X11 X12 X13

wherein

X1 is L, C, I, Y, V, W or F (preferably L, C, I or F, especiallypreferably L);

X2 is K, R, H, E, D, C, V, A, I, Q, S, T or L (preferably K, R, D or E);

X3 is Q, D, E, A, S, I, F, K, R, L, M, T, G, N, W or V (preferably Q, D,E, A, S, I, V, especially preferably Q);

X4 is N, D, E, S, A, M, K, R, G, T, W or Q (preferably N, D, E or S);

X5 is Q, D, E, M, F, I, S, K, R, C, W or Y (preferably Q, D, E, F, I orM);

X6 is S, D, M, N, E, I, A, R, F, H, W, K, L, Y, Q or G (preferably S, M,E or D);

X7 is Q, D, E, I, K, R, T, V, F, N, S, L, W or M (preferably Q, M, E orD);

X8 is I, A, S, L, D, E, B or V;

X9 is K, C, D, E, R, A, M, T, W, H, Q or Y (preferably K or R);

X10 is E, D, R, Q or K (preferably E or D);

X11 is A, C, I, F, L, G, H or V (preferably A);

X12 is T, C, L, F, I, V, M, K, R or W (preferably T, L or W); and

X13 is E, D, N, V, Y, K, A, F, G, H, I, Q, L, M, R, S, T or W(preferably E, D, R, K or W),

or a peptidomimetic or analogue thereof. Additionally X5 may be L or T,X9 may be L or S.

The term “RIAD polypeptide” is also meant to include a polypeptidecomprising a PKA I anchoring disrupting molecule or AKAP mimic asdefined in U.S. Patent Application Publication No. 20080248008, which ishereby incorporated by reference in its entirety. In general, themolecule or mimic is a polypeptide. A “RIAD polypeptide” binds PKA RI asassessed according to the KD between the RIAD polypeptide and thebinding site of the PKA RI molecule as described in U.S. PatentApplication No. 20080248008. Preferably the KD should be 0.01-500 nM,preferably 0.01-10 nM when assessed in vitro. The dissociation constants(KD) may be measured directly by fluorescence polarization as describedin U.S. Patent Application No. 20080248008. In certain embodiments, theterm “RIAD polypeptide” also includes a polypeptide having a RISRsubunit (e.g., the amino acid sequence of SEQ ID NO: 64, or derivativethereof).

The term “RISR subunit” is meant to include a polypeptide having anamino acid sequence of SEQ ID NO: 64, or a derivative thereof. Forexample, the term “RISR subunit” includes polypeptides having greaterthan 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%) sequence identity to SEQID NO: 64. Additionally, or alternatively, the term “RISR subunit”includes polypeptides having the sequence of SEQ ID NO: 64 having one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) conservative aminoacid substitutions (as defined herein).

The term “Ezrin polypeptide” is defined as a polypeptide comprising aPKA I anchoring disrupting molecule having an amino acid sequence of SEQID NO: 108 (KSQEQLAAELAEYTAKIALL), or a derivative thereof. As usedherein, neither the term “Ezrin polypeptide” nor a polypeptide orprotein comprising an Ezrin polypeptide, includes full length, naturallyoccurring human Ezrin (i.e., as set forth in GenBank accession numberAAH68458.1), nor do they include a polypeptide having more than 120consecutive amino acids with greater than 95% identity to naturallyoccurring human Ezrin. For example, the term “Ezrin polypeptide” is apolypeptide having at least 80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%)sequence identity to SEQ ID NO: 108. Additionally, or alternatively, theterm “Ezrin polypeptide” includes polypeptides having the sequence ofSEQ ID NO: 108 having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more) conservative amino acid substitutions (as defined herein).

The term “Ezrin-derived RISR subunit” meant to include a polypeptidehaving an amino acid sequence of SEQ ID NO: 109(MQMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEADRMAALRAKEELERQAVDQI), or a derivative thereof. For example, the term“Ezrin-derived RISR subunit” includes polypeptides having greater than80% (e.g., 85%, 90%, 95%, 97%, 98%, 99%) sequence identity to SEQ ID NO:109. Additionally, or alternatively, the term “Ezrin-derived RISRsubunit” includes polypeptides having the sequence of SEQ ID NO: 109having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)conservative amino acid substitutions (as defined herein).

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa set of polypeptides, typically two in the simplest embodiments, whichwhen in an immune effector cell, provides the cell with specificity fora target cell, typically a cancer cell, and with intracellular signalgeneration. In some embodiments, a CAR comprises at least anextracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined below. In some aspects, the set of polypeptides arecontiguous with each other. In some embodiments, the set of polypeptidesinclude a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signaling domain. In oneaspect, the stimulatory molecule is the zeta chain associated with the Tcell receptor complex. In one aspect, the cytoplasmic signaling domainfurther comprises one or more functional signaling domains derived fromat least one costimulatory molecule as defined below. In one aspect, thecostimulatory molecule is chosen from the costimulatory moleculesdescribed herein, e.g., 4-1BB (i.e., CD137), CD27 and/or CD28. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising a functional signaling domainderived from a stimulatory molecule. In one aspect, the CAR comprises achimeric fusion protein comprising an extracellular antigen bindingdomain, a transmembrane domain and an intracellular signaling domaincomprising a functional signaling domain derived from a costimulatorymolecule and a functional signaling domain derived from a stimulatorymolecule. In one aspect, the CAR comprises a chimeric fusion proteincomprising an extracellular antigen binding domain, a transmembranedomain and an intracellular signaling domain comprising two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more costimulatory molecule(s) anda functional signaling domain derived from a stimulatory molecule. Inone aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen binding domain, wherein the leader sequence isoptionally cleaved from the antigen binding domain (e.g., a scFv) duringcellular processing and localization of the CAR to the cellularmembrane.

A CAR that comprises an antigen binding domain (e.g., a scFv, or TCR)that targets a specific tumor maker X, such as those described herein,is also referred to as XCAR. For example, a CAR that comprises anantigen binding domain that targets CD19 is referred to as CD19CAR.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of anantibody, that retains the ability to specifically interact with (e.g.,by binding, steric hindrance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(seeU.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked, e.g., via a synthetic linker, e.g., a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv. The precise amino acid sequence boundaries of a given CDR can bedetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme), or a combination thereof.

As used herein, the term “binding domain” or “antibody molecule” refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody, or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “anti-cancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of cancer cells, adecrease in the number of metastases, an increase in life expectancy,decrease in cancer cell proliferation, decrease in cancer cell survival,or amelioration of various physiological symptoms associated with thecancerous condition.

An “anti-cancer effect” can also be manifested by the ability of thepeptides, polynucleotides, cells and antibodies in prevention of theoccurrence of cancer in the first place. The term “anti-tumor effect”refers to a biological effect which can be manifested by various means,including but not limited to, e.g., a decrease in tumor volume, adecrease in the number of tumor cells, a decrease in tumor cellproliferation, or a decrease in tumor cell survival.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” refers to a disease characterized by the uncontrolledgrowth of aberrant cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. Examples ofvarious cancers are described herein and include but are not limited to,breast cancer, prostate cancer, ovarian cancer, cervical cancer, skincancer, pancreatic cancer, colorectal cancer, renal cancer, livercancer, brain cancer, lymphoma, leukemia, lung cancer and the like. Theterms “tumor” and “cancer” are used interchangeably herein, e.g., bothterms encompass solid and liquid, e.g., diffuse or circulating, tumors.As used herein, the term “cancer” or “tumor” includes premalignant, aswell as malignant cancers and tumors.

“Derived from” as that term is used herein, indicates a relationshipbetween a first and a second molecule. It generally refers to structuralsimilarity between the first molecule and a second molecule and does notconnotate or include a process or source limitation on a first moleculethat is derived from a second molecule. For example, in the case of anintracellular signaling domain that is derived from a CD3zeta molecule,the intracellular signaling domain retains sufficient CD3zeta structuresuch that is has the required function, namely, the ability to generatea signal under the appropriate conditions. It does not connotate orinclude a limitation to a particular process of producing theintracellular signaling domain, e.g., it does not mean that, to providethe intracellular signaling domain, one must start with a CD3zetasequence and delete unwanted sequence, or impose mutations, to arrive atthe intracellular signaling domain.

The phrase “disease associated with expression of a tumor antigen asdescribed herein” includes, but is not limited to, a disease associatedwith expression of a tumor antigen as described herein or conditionassociated with cells which express a tumor antigen as described hereinincluding, e.g., proliferative diseases such as a cancer or malignancyor a precancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia; or a noncancer related indication associatedwith cells which express a tumor antigen as described herein. In oneaspect, a cancer associated with expression of a tumor antigen asdescribed herein is a hematological cancer. In one aspect, a cancerassociated with expression of a tumor antigen as described herein is asolid cancer. Further diseases associated with expression of a tumorantigen described herein include, but not limited to, e.g., atypicaland/or non-classical cancers, malignancies, precancerous conditions orproliferative diseases associated with expression of a tumor antigen asdescribed herein. Non-cancer related indications associated withexpression of a tumor antigen as described herein include, but are notlimited to, e.g., autoimmune disease, (e.g., lupus), inflammatorydisorders (allergy and asthma) and transplantation. In some embodiments,the tumor antigen-expressing cells express, or at any time expressed,mRNA encoding the tumor antigen. In an embodiment, the tumorantigen-expressing cells produce the tumor antigen protein (e.g.,wild-type or mutant), and the tumor antigen protein may be present atnormal levels or reduced levels. In an embodiment, the tumorantigen-expressing cells produced detectable levels of a tumor antigenprotein at one point, and subsequently produced substantially nodetectable tumor antigen protein.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions are onesin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, one or more amino acid residues within a CAR of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered CAR can be tested using the functionalassays described herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with itscognate ligand (or tumor antigen in the case of a CAR) thereby mediatinga signal transduction event, such as, but not limited to, signaltransduction via the TCR/CD3 complex or signal transduction via theappropriate NK receptor or signaling domains of the CAR. Stimulation canmediate altered expression of certain molecules.

The term “stimulatory molecule,” refers to a molecule expressed by animmune cell (e.g., T cell, NK cell, B cell) that provides thecytoplasmic signaling sequence(s) that regulate activation of the immunecell in a stimulatory way for at least some aspect of the immune cellsignaling pathway. In one aspect, the signal is a primary signal that isinitiated by, for instance, binding of a TCR/CD3 complex with an MHCmolecule loaded with peptide, and which leads to mediation of a T cellresponse, including, but not limited to, proliferation, activation,differentiation, and the like. A primary cytoplasmic signaling sequence(also referred to as a “primary signaling domain”) that acts in astimulatory manner may contain a signaling motif which is known asimmunoreceptor tyrosine-based activation motif or ITAM. Examples of anITAM containing cytoplasmic signaling sequence that is of particular usein the invention includes, but is not limited to, those derived from CD3zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc EpsilonR1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.In a specific CAR of the invention, the intracellular signaling domainin any one or more CARS of the invention comprises an intracellularsignaling sequence, e.g., a primary signaling sequence of CD3-zeta. In aspecific CAR of the invention, the primary signaling sequence ofCD3-zeta is the sequence provided as SEQ ID NO:18, or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like. In a specific CAR of the invention, the primary signalingsequence of CD3-zeta is the sequence as provided in SEQ ID NO:20, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain generates a signal that promotes an immune effector function ofthe CAR containing cell, e.g., a CART cell. Examples of immune effectorfunction, e.g., in a CART cell, include cytolytic activity and helperactivity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CART, a primary intracellularsignaling domain can comprise a cytoplasmic sequence of a T cellreceptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBank Acc. No. BAG36664.1, orthe equivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain, or functional derivatives thereof, that aresufficient to functionally transmit an initial signal necessary for Tcell activation. In one aspect the cytoplasmic domain of zeta comprisesresidues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like, that are functional orthologs thereof. In one aspect, the“zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is thesequence provided as SEQ ID NO:18. In one aspect, the “zeta stimulatorydomain” or a “CD3-zeta stimulatory domain” is the sequence provided asSEQ ID NO:20.

The term a “costimulatory molecule” refers to a cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arecontribute to an efficient immune response. Costimulatory moleculesinclude, but are not limited to an MHC class I molecule, BTLA and a Tollligand receptor, as well as OX40, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of suchcostimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain can be the intracellularportion of a costimulatory molecule. A costimulatory molecule can berepresented in the following protein families: TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), and activating NK cellreceptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137),OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT,NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and aligand that specifically binds with CD83, and the like.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment orderivative thereof.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:14 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include Tcells, e.g., alpha/beta T cells and gamma/delta T cells, B cells,natural killer (NK) cells, natural killer T (NKT) cells, mast cells, andmyeloid-derived phagocytes.

“Immune effector function or immune effector response,” as that term isused herein, refers to function or response, e.g., of an immune effectorcell, that enhances or promotes an immune attack of a target cell. E.g.,an immune effector function or response refers a property of a T or NKcell that promotes killing or the inhibition of growth or proliferation,of a target cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody orantibody fragment, where the whole molecule is of human origin orconsists of an amino acid sequence identical to a human form of theantibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The terms “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically a protein, carbohydrate or lipid) thatis expressed on the surface of a cancer cell, either entirely or as afragment (e.g., MHC/peptide), and which is useful for the preferentialtargeting of a pharmacological agent to the cancer cell. In someembodiments, a tumor antigen is a marker expressed by both normal cellsand cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In someembodiments, a tumor antigen is a cell surface molecule that isoverexpressed in a cancer cell in comparison to a normal cell, forinstance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a tumor antigen is a cell surface molecule that isinappropriately synthesized in the cancer cell, for instance, a moleculethat contains deletions, additions or mutations in comparison to themolecule expressed on a normal cell. In some embodiments, a tumorantigen will be expressed exclusively on the cell surface of a cancercell, entirely or as a fragment (e.g., MHC/peptide), and not synthesizedor expressed on the surface of a normal cell. In some embodiments, theCARs of the present invention includes CARs comprising an antigenbinding domain (e.g., antibody or antibody fragment) that binds to a MHCpresented peptide. Normally, peptides derived from endogenous proteinsfill the pockets of Major histocompatibility complex (MHC) class Imolecules, and are recognized by T cell receptors (TCRs) on CD8+ Tlymphocytes. The MHC class I complexes are constitutively expressed byall nucleated cells. In cancer, virus-specific and/or tumor-specificpeptide/MHC complexes represent a unique class of cell surface targetsfor immunotherapy. TCR-like antibodies targeting peptides derived fromviral or tumor antigens in the context of human leukocyte antigen(HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., JVirol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165;Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci TranslMed 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 201219(2):84-100). For example, TCR-like antibody can be identified fromscreening a library, such as a human scFv phage displayed library.

The term “tumor-supporting antigen” or “cancer-supporting antigen”interchangeably refer to a molecule (typically a protein, carbohydrateor lipid) that is expressed on the surface of a cell that is, itself,not cancerous, but supports the cancer cells, e.g., by promoting theirgrowth or survival e.g., resistance to immune cells. Exemplary cells ofthis type include stromal cells and myeloid-derived suppressor cells(MDSCs). The tumor-supporting antigen itself need not play a role insupporting the tumor cells so long as the antigen is present on a cellthat supports cancer cells.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence(Gly-Gly-Gly-Ser)_(n), where n is a positive integer equal to or greaterthan 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 andn=10 (SEQ ID NO:28). In one embodiment, the flexible polypeptide linkersinclude, but are not limited to, (Gly₄ Ser)₄ (SEQ ID NO:29) or (Gly₄Ser)₃ (SEQ ID NO:30). In another embodiment, the linkers includemultiple repeats of (Gly₂Ser), (GlySer) or (Gly₃Ser) (SEQ ID NO:31).Also included within the scope of the invention are linkers described inWO2012/138475, incorporated herein by reference).

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000 (SEQ ID NO:34), preferably greater than 64, more preferably greater than 100, mostpreferably greater than 300 or 400. poly(A) sequences can be modifiedchemically or enzymatically to modulate mRNA functionality such aslocalization, stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a proliferative disorder, or the amelioration of one or moresymptoms (preferably, one or more discernible symptoms) of aproliferative disorder resulting from the administration of one or moretherapies (e.g., one or more therapeutic agents such as a CAR of theinvention). In specific embodiments, the terms “treat”, “treatment” and“treating” refer to the amelioration of at least one measurable physicalparameter of a proliferative disorder, such as growth of a tumor, notnecessarily discernible by the patient. In other embodiments the terms“treat”, “treatment” and “treating”-refer to the inhibition of theprogression of a proliferative disorder, either physically by, e.g.,stabilization of a discernible symptom, physiologically by, e.g.,stabilization of a physical parameter, or both. In other embodiments theterms “treat”, “treatment” and “treating” refer to the reduction orstabilization of tumor size or cancerous cell count.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer,non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer,cervical cancer, bladder cancer, kidney cancer and adenocarcinomas suchas breast cancer, prostate cancer, ovarian cancer, pancreatic cancer,and the like.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a binding partner (e.g., a tumor antigen)protein present in a sample, but which antibody or ligand does notsubstantially recognize or bind other molecules in the sample.

“Regulatable chimeric antigen receptor (RCAR),” as that term is usedherein, refers to a set of polypeptides, typically two in the simplestembodiments, which when in a RCARX cell, provides the RCARX cell withspecificity for a target cell, typically a cancer cell, and withregulatable intracellular signal generation or proliferation, which canoptimize an immune effector property of the RCARX cell. An RCARX cellrelies at least in part, on an antigen binding domain to providespecificity to a target cell that comprises the antigen bound by theantigen binding domain. In an embodiment, an RCAR includes adimerization switch that, upon the presence of a dimerization molecule,can couple an intracellular signaling domain to the antigen bindingdomain.

“Membrane anchor” or “membrane tethering domain”, as that term is usedherein, refers to a polypeptide or moiety, e.g., a myristoyl group,sufficient to anchor an extracellular or intracellular domain to theplasma membrane.

“Switch domain,” as that term is used herein, e.g., when referring to anRCAR, refers to an entity, typically a polypeptide-based entity, that,in the presence of a dimerization molecule, associates with anotherswitch domain. The association results in a functional coupling of afirst entity linked to, e.g., fused to, a first switch domain, and asecond entity linked to, e.g., fused to, a second switch domain. A firstand second switch domain are collectively referred to as a dimerizationswitch. In embodiments, the first and second switch domains are the sameas one another, e.g., they are polypeptides having the same primaryamino acid sequence, and are referred to collectively as ahomodimerization switch. In embodiments, the first and second switchdomains are different from one another, e.g., they are polypeptideshaving different primary amino acid sequences, and are referred tocollectively as a heterodimerization switch. In embodiments, the switchis intracellular. In embodiments, the switch is extracellular. Inembodiments, the switch domain is a polypeptide-based entity, e.g., FKBPor FRB-based, and the dimerization molecule is small molecule, e.g., arapalogue. In embodiments, the switch domain is a polypeptide-basedentity, e.g., an scFv that binds a myc peptide, and the dimerizationmolecule is a polypeptide, a fragment thereof, or a multimer of apolypeptide, e.g., a myc ligand or multimers of a myc ligand that bindto one or more myc scFvs. In embodiments, the switch domain is apolypeptide-based entity, e.g., myc receptor, and the dimerizationmolecule is an antibody or fragments thereof, e.g., myc antibody.

“Dimerization molecule,” as that term is used herein, e.g., whenreferring to an RCAR, refers to a molecule that promotes the associationof a first switch domain with a second switch domain. In embodiments,the dimerization molecule does not naturally occur in the subject, ordoes not occur in concentrations that would result in significantdimerization. In embodiments, the dimerization molecule is a smallmolecule, e.g., rapamycin or a rapalogue, e.g., RAD001.

The term “bioequivalent” refers to an amount of an agent other than thereference compound (e.g., RAD001), required to produce an effectequivalent to the effect produced by the reference dose or referenceamount of the reference compound (e.g., RAD001). In an embodiment theeffect is the level of mTOR inhibition, e.g., as measured by P70 S6kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay,e.g., as measured by an assay described herein, e.g., the Boulay assay.In an embodiment, the effect is alteration of the ratio of PD-1positive/PD-1 negative T cells, as measured by cell sorting. In anembodiment a bioequivalent amount or dose of an mTOR inhibitor is theamount or dose that achieves the same level of P70 S6 kinase inhibitionas does the reference dose or reference amount of a reference compound.In an embodiment, a bioequivalent amount or dose of an mTOR inhibitor isthe amount or dose that achieves the same level of alteration in theratio of PD-1 positive/PD-1 negative T cells as does the reference doseor reference amount of a reference compound.

The term “low, immune enhancing, dose” when used in conjunction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing, dose of mTOR inhibitor results ina decrease in the number of PD-1 positive T cells and/or an increase inthe number of PD-1 negative T cells, or an increase in the ratio of PD-1negative T cells/PD-1 positive T cells. In an embodiment, the low,immune enhancing, dose of mTOR inhibitor results in an increase in thenumber of naive T cells. In an embodiment, the low, immune enhancing,dose of mTOR inhibitor results in one or more of the following:

an increase in the expression of one or more of the following markers:CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on memory T cells,e.g., memory T cell precursors; a decrease in the expression of KLRG1,e.g., on memory T cells, e.g., memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62L^(high) increased CD127^(high), increased CD27⁺, decreased KLRG1,and increased BCL2;

wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject.

“Refractory” as used herein refers to a disease, e.g., cancer, that doesnot respond to a treatment. In embodiments, a refractory cancer can beresistant to a treatment before or at the beginning of the treatment. Inother embodiments, the refractory cancer can become resistant during atreatment. A refractory cancer is also called a resistant cancer.

“Relapsed” as used herein refers to the return of a disease (e.g.,cancer) or the signs and symptoms of a disease such as cancer after aperiod of improvement, e.g., after prior treatment of a therapy, e.g.,cancer therapy

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

DESCRIPTION

Provided herein are compositions of matter and methods of use for thetreatment of a disease such as cancer using immune effector cells (e.g.,T cells, NK cells) engineered with

CARs of the invention.

In one aspect, the invention provides a number of chimeric antigenreceptors (CAR) comprising an antigen binding domain (e.g., antibody orantibody fragment, TCR or TCR fragment) engineered for specific bindingto a tumor antigen, e.g., a tumor antigen described herein. In oneaspect, the invention provides an immune effector cell (e.g., T cell, NKcell) engineered to express a CAR, wherein the engineered immuneeffector cell exhibits an anticancer property. In one aspect, a cell istransformed with the CAR and the CAR is expressed on the cell surface.In some embodiments, the cell (e.g., T cell, NK cell) is transduced witha viral vector encoding a CAR. In some embodiments, the viral vector isa retroviral vector. In some embodiments, the viral vector is alentiviral vector. In some such embodiments, the cell may stably expressthe CAR. In another embodiment, the cell (e.g., T cell, NK cell) istransfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR.In some such embodiments, the cell may transiently express the CAR.

In one aspect, the antigen binding domain of a CAR described herein is ascFv antibody fragment. In one aspect, such antibody fragments arefunctional in that they retain the equivalent binding affinity, e.g.,they bind the same antigen with comparable affinity, as the IgG antibodyfrom which it is derived. In other embodiments, the antibody fragmenthas a lower binding affinity, e.g., it binds the same antigen with alower binding affinity than the antibody from which it is derived, butis functional in that it provides a biological response describedherein. In one embodiment, the CAR molecule comprises an antibodyfragment that has a binding affinity KD of 10⁻⁴ M to 10⁻⁸ M, e.g., 10⁻⁵M to 10⁻⁷ M, e.g., 10⁻⁶ M or 10⁻⁷ M, for the target antigen. In oneembodiment, the antibody fragment has a binding affinity that is atleast five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or1,000-fold less than a reference antibody, e.g., an antibody describedherein.

In one aspect such antibody fragments are functional in that theyprovide a biological response that can include, but is not limited to,activation of an immune response, inhibition of signal-transductionorigination from its target antigen, inhibition of kinase activity, andthe like, as will be understood by a skilled artisan.

In one aspect, the antigen binding domain of the CAR is a scFv antibodyfragment that is humanized compared to the murine sequence of the scFvfrom which it is derived.

In one aspect, the antigen binding domain of a CAR of the invention(e.g., a scFv) is encoded by a nucleic acid molecule whose sequence hasbeen codon optimized for expression in a mammalian cell. In one aspect,entire CAR construct of the invention is encoded by a nucleic acidmolecule whose entire sequence has been codon optimized for expressionin a mammalian cell. Codon optimization refers to the discovery that thefrequency of occurrence of synonymous codons (i.e., codons that code forthe same amino acid) in coding DNA is biased in different species. Suchcodon degeneracy allows an identical polypeptide to be encoded by avariety of nucleotide sequences. A variety of codon optimization methodsis known in the art, and include, e.g., methods disclosed in at leastU.S. Pat. Nos. 5,786,464 and 6,114,148.

In one aspect, the CARs of the invention combine an antigen bindingdomain of a specific antibody with an intracellular signaling molecule.For example, in some aspects, the intracellular signaling moleculeincludes, but is not limited to, CD3-zeta chain, 4-1BB and CD28signaling modules and combinations thereof. In one aspect, the antigenbinding domain binds to a tumor antigen as described herein.

Furthermore, the present invention provides CARs and CAR-expressingcells and their use in medicaments or methods for treating, among otherdiseases, cancer or any malignancy or autoimmune diseases involvingcells or tissues which express a tumor antigen as described herein.

In one aspect, the CAR of the invention can be used to eradicate anormal cell that express a tumor antigen as described herein, therebyapplicable for use as a cellular conditioning therapy prior to celltransplantation. In one aspect, the normal cell that expresses a tumorantigen as described herein is a normal stem cell and the celltransplantation is a stem cell transplantation.

In one aspect, the invention provides an immune effector cell (e.g., Tcell, NK cell) engineered to express a chimeric antigen receptor (CAR),wherein the engineered immune effector cell exhibits an antitumorproperty. A preferred antigen is a cancer associated antigen (i.e.,tumor antigen) described herein. In one aspect, the antigen bindingdomain of the CAR comprises a partially humanized antibody fragment. Inone aspect, the antigen binding domain of the CAR comprises a partiallyhumanized scFv. Accordingly, the invention provides CARs that comprisesa humanized antigen binding domain and is engineered into a cell, e.g.,a T cell or a NK cell, and methods of their use for adoptive therapy.

In one aspect, the CARs of the invention comprise at least oneintracellular domain selected from the group of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD27 signal domain, aCD3zeta signal domain, and any combination thereof. In one aspect, theCARs of the invention comprise at least one intracellular signalingdomain is from one or more costimulatory molecule(s) other than a CD137(4-1BB) or CD28.

Sequences of some examples of various components of CARs of the instantinvention is listed in Table 1A, where aa stands for amino acids, and nastands for nucleic acids that encode the corresponding peptide.

TABLE 1A Sequences of various components of CAR (aa—amino acids,na—nucleic acids that encodes the corresponding protein) SEQ Corresp. IDTo NO description Sequence huCD19 1 EF-1CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA 100 promoterTCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 2 Leader (aa) MALPVTALLLPLALLLHAARP13 3 Leader (na) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCT 54CTGCTGCTGCATGCCGCTAGACCC 4 CD 8 hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 14 (aa) FACD 5 CD8 hingeACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCC 55 (na)CACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAG GGGGCTGGACTTCGCCTGTGAT 6 Ig4 hingeESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 102 (aa)CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM7 Ig4 hinge GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGC 103 (na)CCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGG GCAAGATG 8 IgD hingeRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 47 (aa)GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH 9 IgD hingeAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGT 48 (na)TCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGAC TGACCATT 10 GS GGGGSGGGGS 49hinge/linker (aa) 11 GS GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 hinge/linker(na) 12 CD8TM (aa) IYIWAPLAGTCGVLLLSLVITLYC 15 13 CD8 TMATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGT 56 (na)CCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 14 4-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC 16 intracellular EL domain (aa)15 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA 60 intracellularACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG domain (na)ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTG 16 CD27 (aa)QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRK 51 PEPACSP 17 CD27 (na)AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACAT 52GAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCT ATCGCTCC 18 CD3-zetaRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRR 17 (aa)GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 19 CD3-zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA 101 (na)CAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 20 CD3-zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR 43 (aa)GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 21 CD3-zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTA 44 (na) CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 22 linker GGGGS 18 23 linkerGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 24 PD-1Pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaextracellularafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslrael domain(aa) rvterraeyptahpspsprpagqfqtlv 25 PD-1Cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactextracellularcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatca domain(na) ttcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtc 26 PD-1 CARMalpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntses (aa)with fvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtyl signalcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkagrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr 27 PD-1 CARAtggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacc (na)acccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc 28 linker (Gly-Gly-Gly-Ser)_(n), where n= 1-10 105 29 linker (Gly4 Ser)4 106 30 linker (Gly4 Ser)3 107 31 linker(Gly3Ser) 108 32 polyA aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 118 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 33 polyA aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 104 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 34 polyA aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 109 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 35 polyAtttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 110tttttttttt tttttttttt tttttttttt tttttttttt 36 polyA tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 111 tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 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tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttttttttttttt 37 polyA aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 112 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 38 polyA aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 113 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 39 PD1 CARPgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdkla (aa)afpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydal hmqalppr

RIAD Peptides

Adenosine and prostaglandin E2 are among the most important ofimmunosuppressive factors that may interfere with CAR mediated killingof tumors (e.g., solid tumors). Both these ligands, through their ownG-coupled receptors, trigger the production of cyclic AMP (cAMP) whichin turn can activate the enzyme protein kinase A, or PKA. It is wellestablished that prolonged cAMP signaling induces PKA activity, whichthen affects multiple proteins in the T cell signaling cascade. One ofthe most important and proximal effects is activation of the kinase Cskwhich then inhibits Lck. This results in inhibition of T cell signalingwhich blocks T cell receptor (TCR)-induced T cell proliferation andcytotoxic activity.

In order for PKA to elicit its functions, it must be tethered to lipidrafts in close proximity to adenylyl cyclase, the enzyme thatmetabolizes ATP to generate cAMP. The tethering and subsequentcompartmentalization of PKA and its effects are mediated by A-kinaseanchoring proteins (AKAP). AKAP therefore serve as a platform on whichcAMP and PKA signaling converge, and provide temporal and spatialregulation of these entities. With regard to T cell signaling PKA mustbe directed to the TCR by binding to a protein called ezrin whichinserts into the membrane and serves as the AKAP.

The PKA holoenzyme is a heterotetramer consisting of 2 regulatorysubunits (RI and RII) and 2 catalytic subunits (CI and CII). Uponactivation by cAMP, the R subunits dissociate, and the C subunitsproceed to phosphorylate a large myriad of target substrates; thecAMP-PKA signaling cascade is one of the most ubiquitous andwell-established second messenger systems to date. Within the tumormicroenvironment, suppressive factors such as prostaglandin E2 (PGE2),adenosine, and other cAMP-activating ligands may neutralize T cellability to kill tumors. The invention features the attenuation of cAMPsignaling in T cells, and more specifically CAR T cells, to prolongTCR-mediated signaling and better killing capacity.

RIAD binds to the RI subunit of PKA with high affinity and disrupts PKAanchoring to ezrin, thereby neutralizing PKA signaling (Carlson C, etal. J Biol Chem 281: 30, 2006). FIG. 22 shows how this works. Normally(left panel of FIG. 22), cAMP binds to PKA which activates it. The PKAbinds to ezrin and is brought in contact with Csk. Csk is activatedwhich then phosphorylates and inactivates Lck which stops TCR signaling.With RIAD present (right panel of FIG. 22), the PKA-cAMP complex cannotlocalize to ezrin and, thus, does not have access to Csk. Carlson et al.reported that the RIAD-mediated displacement of PKA ultimatelydiminished phosphorylation of Tyr-505 on Lck, and hence upregulated TCRsignaling.

RISR (RI specifier region) is a polypeptide that enhances RIAD bindingto PKA, thereby augmenting the release of T cell inhibition by cAMP.

The sequences of RIAD, RISR, RISR-RIAD, RIAD-T2A, and RISR-RIAD-T2A areset forth below.

TABLE 1B RIAD and RISR sequences Gene Nucleic acid sequence Amino acidsequence RIAD ctggaacagtatgcgaaccagctggcggatcagattattaLEQYANQLADQIIKEATE aagaagcgaccgaa (SEQ ID NO: 68) (SEQ ID NO: 63) RISRgaaagcaaacgccaggaagaagcggaacagcgcaaa ESKRRQEEAEQRK (SEQ (SEQ ID NO: 69)ID NO: 64) RISR-RIAD gaaagcaaacgccaggaagaagcggaacagcgcaaa ESKRRQEEAEQRK-ctggaacagtatgcgaaccagctggcggatcagattatta LEQYANQLADQIIKEATEaagaagcgaccgaa (SEQ ID NO: 70) (SEQ ID NO: 65) RIAD-T2Actggaacagtatgcgaaccagctggcggatcagattatta Leqyanqladqiikeatetrtrpleqkliaagaagcgaccgaaacgcgtacgcggccgctcgagca seedlaandildykddddkgsgegrggaaactcatctcagaagaggatctggcagcaaatgatatc slltcgdveenpg (SEQ ID NO:ctggattacaaggatgacgacgataagggcagcggaga 66)gggcagaggaagtcttctaacatgcggtgacgtggagg agaatcccggc (SEQ ID NO: 71)RISR-RIAD- ccaccatggaaagcaaacgccgccaggaagaagcggaEskrrqeeaeqrkleqyanqladqiik T2A acagcgcaaactggaacagtatgcgaaccagctggcggeatetrtrpleqkliseedlaandildyk atcagattattaaagaagcgaccgaaacgcgtacgcggcddddkgsgegrgslltcgdveenpg cgctcgagcagaaactcatctcagaagaggatctggcag (SEQID NO: 67) caaatgatatcctggattacaaggatgacgacgataagggcagcggagagggcagaggaagtcttctaacatgcggt gacgtggaggagaatcccggc (SEQ ID NO:79)

In some embodiments, the RIAD sequence comprises LEQYANQLADQIIKEATE (SEQID NO: 63), conservative changes to the sequence, homologous sequencesfrom related proteins, and fragments thereof. In another embodiment, anucleic acid sequence encoding the RIAD sequence comprises5′-GTCGACCTGGAGCAGTACGCCAACCAGCTGGCCGACCAGATCATCAAGGAGGCCACCGAGGGATCC-3′ (SEQ ID NO: 113), conservative changes to the sequence,homologous sequences from related genes, and fragments thereof.

Ezrin polypeptides include, e.g., SEQ ID NO: 108 and, optionally, SEQ IDNO: 109. In one embodiment, the Ezrin polypeptide comprises the aminoacid sequence of MQMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEADRMAALRAKEELERQAVDQIKSQEQLAAELAEYTAKIALL (SEQ ID NO: 110). The Ezrinpolypeptide can be encoded by, e.g., a nucleic acid having the sequenceset forth in SEQ ID NO: 111(ATGCAGATGATGCGCGAAAAAGAAGAACTGATGCTGCGCCTGCAGGATTATGAAGAAAAAACCAAAAAAGCGGAACGCGAACTGAGCGAACAGATTCAGCGCGCGCTGCAGCTGGAAGAAGAACGCAAACGCGCGCAGGAAGAAGCGGAACGCCTGGAAGCGGATCGCATGGCGGCGCTGCGCGCGAAAGAAGAACTGGAACGCCAGGCGGTGGATCAGATTAAAAGCCAGGAACAGCTGGCGGCGGAACTGGCGGAATATACCGCGAAAATTGCGCTGCTG).

In some embodiments, a peptide (RIAD) further comprises a region toenhance specificity to PKA; e.g., the regulatory subunit I (RI)specifier region, or RISR. In one embodiment, the peptide comprises atleast one domain selected from the group consisting of a regulatorysubunit I anchoring disruptor (RIAD), a regulatory subunit I specifierregion (RISR), and a combination thereof.

In some embodiments, the peptide binds a PKA, an A-kinase anchoringprotein (AKAP) (such as Ezrin), or another molecule that disrupts PKAand AKAP binding. In one embodiment, the peptide binds a regulatorysubunit of PKA. In an embodiment, the peptide comprises an amphipathichelix domain. In another embodiment, the peptide comprises a fragment ofan AKAP, wherein the fragment comprises an amphipathic helix domain ofthe AKAP.

In an embodiment, the peptide comprises a cluster of amino acids withbasic side chains. The peptide including a cluster of basic amino acidsis capable of binding to a regulatory subunit of PKA. In one embodiment,the cluster of basic amino acids binds PKA. In another embodiment, thepeptide comprises a fragment of an AKAP, wherein the fragment comprisesat least one cluster of basic amino acids of the AKAP.

In another embodiment, the peptide comprises an amphipathic helix domainand at least one cluster of basic amino acids. In one embodiment, thepeptide comprises at least one fragment of an AKAP, wherein the fragmentcomprises an amphipathic helix domain and at least one cluster of basicamino acids from the AKAP. The peptide including the amphipathic helixdomain and cluster of basic amino acids is capable of binding to aregulatory subunit of PKA. In one embodiment, the amphipathic helixdomain and the cluster of basic amino acids bind PKA. In embodiments,the inclusion of both an amphipathic helix domain and at least onecluster of basic amino acids enhances binding to the regulatory subunitof PKA. The enhanced binding can include higher binding efficiency,higher affinity for PKA, greater specificity for PKA, etc.

In another embodiment, the peptide is capable of binding an amphipathichelix domain. In one embodiment, the peptide comprises a fragment ofPKA, wherein the fragment comprises a PKA domain that binds to anamphipathic helix domain. In such an embodiment, the peptide is capableof binding the amphipathic helix domain of the AKAP, thereby disruptingPKA binding to the AKAP.

In still another embodiment, the peptide binds one or more othermolecules to disrupt PKA and AKAP binding.

In embodiments, the peptide has a length of about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60, or more amino acids. In oneembodiment, the peptide has a length in the range of about 10 to about60 amino acids. In an exemplary embodiment, the peptide comprises aRIAD. In another embodiment, the peptide comprises an amphipathic helixdomain. The amphipathic helix domain has a length in the range of about10-30 amino acids. In another embodiment, the peptide comprises a RISR.In some embodiments, the peptide comprises at least one cluster of basicamino acids. In other embodiments, the peptide comprising the cluster ofbasic amino acids has a length in the range of about 10 to about 40amino acids. The molar mass of the peptide may be about 5 kD, 6 kD, 7kD, 8 kD, 9 kD, 10 kD, 11 kD, 12 kD, 13 kD, 14 kD, 15 kD, 16 kD, 17 kD,18 kD, 19 kD, 20 kD, 21 kD, 22 kD, 23 kD, 24 kD, 25 kD, 26 kD, 27 kD, 28kD, 29 kD, 30 kD, or more or any molar mass therebetween or less.

In certain embodiments, the peptide includes a RIAD polypeptide orincludes a polypeptide having an amino acid sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityor homology to SEQ ID NO: 63. In certain aspects, the invention includesa nucleic acid sequence encoding the RIAD peptide or includes a nucleicacid encoding a polypeptide having a nucleic acid sequence at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical orhomologous to SEQ ID NO: 68. In certain aspects, the peptide includes aRISR polypeptide or includes a polypeptide having an amino acid sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity or homology to SEQ ID NO: 64. In certain aspects, theinvention includes a nucleic acid sequence encoding the RISR peptide orincludes a nucleic acid sequence encoding a polypeptide having a nucleicacid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical or homologous to a nucleic acid sequence encodingSEQ ID NO: 64 or homologous to SEQ ID NO: 69. In related aspects, thepeptide can be any of the “anchoring disruption molecule or AKAP mimic”disclosed in U.S. Patent Application Publication No. 20080248008, eachof which is hereby specifically incorporated by reference.

In certain embodiments, the present invention features the use of a RIADpolypeptide (e.g., RIAD, RISR, or RISR-RIAD) or Ezrin polypeptide in CARand transgenic TCR-transduced T cells. In certain embodiments, the RIADpolypeptide can be expressed in frame, as a single peptide with the CAR(e.g., at the N terminus of the CAR or at the C terminus of the CAR).The RIAD polypeptide portion or Ezrin polypeptide portion of thisconstruct can be separated from the CAR by a self cleaving sequences(e.g., T2A) or the substrate for an intracellular protease.

Self-cleaving sequences useful in certain embodiments of the inventioninclude those set forth in the following table (in certain embodiments,the residues in parenthesis are optional) (SEQ ID NOS 73-76,respectively, in order of appearance):

TABLE 1C Self-cleaving sequences T2A: (GSG) E G R G S L L T C G D V E EN P G P P2A: (GSG) A T N E S L L K Q A G D V E E N P G P E2A: (GSG) Q CT N Y A L L K L A G D V E S N P G P F2A: (GSG) V K Q T L N F D L L K L AG D V E S N P G P

In other embodiments, the RIAD polypeptide or Ezrin polypeptide can beexpressed as a protein separate from the CAR. In such circumstances, theRIAD polypeptide or Ezrin polypeptide and CAR can be under the controlof separate promoters or under the control of a single promoter, andseparated by an internal ribosomal entry site.

Peptide and Nucleic Acid Compositions

Peptide and nucleic acid compositions are described herein that canattenuate the effect immunosuppressive factors have on T cell activationand signaling. In embodiments, the compositions disrupt PKAlocalization, namely preventing PKA from anchoring to the cell membrane,and enhance the effectiveness of a T cell.

In one aspect, the invention includes a composition comprising a nucleicacid sequence encoding a CAR and a nucleic acid sequence encoding apeptide comprising an amphipathic helix domain and a cluster of basicamino acids, wherein the peptide disrupts PKA and AKAP association. Thenucleic acid encoding the peptide may be a separate molecule from thenucleic acid that encodes the CAR. In this embodiment, the compositioncomprises at least two nucleic acid sequences. The nucleic acidsequences include DNA, RNA, synthetic constructs, and any combinationthereof. The nucleic acid sequences can be included as part of a vector,such as an expression vector or viral vector.

In one embodiment, a single nucleic acid molecule may encode both thepeptide and the CAR, e.g. a bicistronic transcript. In an embodiment,the nucleic acids encoding the peptide are separated from the nucleicacids encoding the CAR by an internal ribosome entry site. In anotherembodiment, the nucleic acid molecule includes a common promoter for thepeptide and the CAR, or separate promoters.

In another embodiment of a single nucleic acid molecule, the nucleicacid sequence may be cleaved, such that the nucleic acids encoding thepeptide are cleaved from the nucleic acids encoding the CAR. In such anembodiment, the nucleic acid sequence further comprises a cleavage site,such as a restriction site, between the nucleic acids encoding thepeptide and the nucleic acids encoding the CAR. The cleavage occursbefore, during, and/or after transcription of the mRNA and beforetranslation into a polypeptide.

In another aspect, a composition comprises a CAR and a peptide thatdisrupts PKA and AKAP binding. The CAR and the peptide may be expressedas a single polypeptide with a cleavage site, such as an enzymatic site,between the two molecules, such that the peptide is cleaved from the CARintracellularly. In this embodiment, during and/or after translation ofthe mRNA encoding the peptide and the CAR, the polypeptide molecule iscleaved, thus the peptide is cleaved from the CAR. The cleavage site maybe an enzymatic site that a proteinase, peptidase, or other enzyme cancleave or an auto-cleavage site between the peptide and the CAR.

In another aspect, the invention includes a composition comprising theCAR and the peptide as separate molecules.

In yet another aspect, the invention includes a composition comprisingone or more vectors (e.g., an expression vector) comprising a nucleicacid sequence encoding the CAR and a nucleic acid sequence encoding thepeptide.

Cancer Associated Antigens

In certain aspects, the present invention provides immune effector cells(e.g., T cells, NK cells) that are engineered to contain one or moreCARs that direct the immune effector cells to cancer. This is achievedthrough an antigen binding domain on the CAR that is specific for acancer associated antigen. There are two classes of cancer associatedantigens (tumor antigens) that can be targeted by the CARs of theinstant invention: (1) cancer associated antigens that are expressed onthe surface of cancer cells; and (2) cancer associated antigens thatitself is intracellular, however, a fragment of such antigen (peptide)is presented on the surface of the cancer cells by MHC (majorhistocompatibility complex).

Accordingly, the present invention provides CARs that target thefollowing cancer associated antigens (tumor antigens): CD19, CD123,CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA,Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3,KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24,PDGFR-beta, PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ireceptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1,sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248,TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid,PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2,TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, MAGE-A1, MAGE A1,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53, p53 mutant, prostein, survivin andtelomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS,SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerasereverse transcriptase, RU1, RU2, intestinal carboxyl esterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A,BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

Tumor-Supporting Antigens

A CAR described herein can comprise an antigen binding domain (e.g.,antibody or antibody fragment, TCR or TCR fragment) that binds to atumor-supporting antigen (e.g., a tumor-supporting antigen as describedherein). In some embodiments, the tumor-supporting antigen is an antigenpresent on a stromal cell or a myeloid-derived suppressor cell (MDSC).Stromal cells can secrete growth factors to promote cell division in themicroenvironment. MDSC cells can inhibit T cell proliferation andactivation. Without wishing to be bound by theory, in some embodiments,the CAR-expressing cells destroy the tumor-supporting cells, therebyindirectly inhibiting tumor growth or survival.

In embodiments, the stromal cell antigen is chosen from one or more of:bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein(FAP) and tenascin. In an embodiment, the FAP-specific antibody is,competes for binding with, or has the same CDRs as, sibrotuzumab. Inembodiments, the MDSC antigen is chosen from one or more of: CD33,CD11b, C14, CD15, and CD66b. Accordingly, in some embodiments, thetumor-supporting antigen is chosen from one or more of: bone marrowstromal cell antigen 2 (BST2), fibroblast activation protein (FAP) ortenascin, CD33, CD11b, C14, CD15, and CD66b.

Chimeric Antigen Receptor (CAR)

The present invention encompasses a recombinant DNA construct comprisingsequences encoding a CAR, wherein the CAR comprises an antigen bindingdomain (e.g., antibody or antibody fragment, TCR or TCR fragment) thatbinds specifically to a cancer associated antigen described herein,wherein the sequence of the antigen binding domain is contiguous withand in the same reading frame as a nucleic acid sequence encoding anintracellular signaling domain. The intracellular signaling domain cancomprise a costimulatory signaling domain and/or a primary signalingdomain, e.g., a zeta chain. The costimulatory signaling domain refers toa portion of the CAR comprising at least a portion of the intracellulardomain of a costimulatory molecule.

In specific aspects, a CAR construct of the invention comprises a scFvdomain, wherein the scFv may be preceded by an optional leader sequencesuch as provided in SEQ ID NO: 2, and followed by an optional hingesequence such as provided in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8or SEQ ID NO:10, a transmembrane region such as provided in SEQ IDNO:12, an intracellular signalling domain that includes SEQ ID NO:14 orSEQ ID NO:16 and a CD3 zeta sequence that includes SEQ ID NO:18 or SEQID NO:20, e.g., wherein the domains are contiguous with and in the samereading frame to form a single fusion protein.

In one aspect, an exemplary CAR constructs comprise an optional leadersequence (e.g., a leader sequence described herein), an extracellularantigen binding domain (e.g., an antigen binding domain describedherein), a hinge (e.g., a hinge region described herein), atransmembrane domain (e.g., a transmembrane domain described herein),and an intracellular stimulatory domain (e.g., an intracellularstimulatory domain described herein). In one aspect, an exemplary CARconstruct comprises an optional leader sequence (e.g., a leader sequencedescribed herein), an extracellular antigen binding domain (e.g., anantigen binding domain described herein), a hinge (e.g., a hinge regiondescribed herein), a transmembrane domain (e.g., a transmembrane domaindescribed herein), an intracellular costimulatory signaling domain(e.g., a costimulatory signaling domain described herein) and/or anintracellular primary signaling domain (e.g., a primary signaling domaindescribed herein).

An exemplary leader sequence is provided as SEQ ID NO: 2. An exemplaryhinge/spacer sequence is provided as SEQ ID NO: 4 or SEQ ID NO:6 or SEQID NO:8 or SEQ ID NO:10. An exemplary transmembrane domain sequence isprovided as SEQ ID NO:12. An exemplary sequence of the intracellularsignaling domain of the 4-1BB protein is provided as SEQ ID NO: 14. Anexemplary sequence of the intracellular signaling domain of CD27 isprovided as SEQ ID NO:16. An exemplary CD3zeta domain sequence isprovided as SEQ ID NO: 18 or SEQ ID NO:20.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises the nucleic acid sequenceencoding an antigen binding domain, e.g., described herein, that iscontiguous with and in the same reading frame as a nucleic acid sequenceencoding an intracellular signaling domain.

In one aspect, the present invention encompasses a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises a nucleic acid sequenceencoding an antigen binding domain, wherein the sequence is contiguouswith and in the same reading frame as the nucleic acid sequence encodingan intracellular signaling domain. An exemplary intracellular signalingdomain that can be used in the CAR includes, but is not limited to, oneor more intracellular signaling domains of, e.g., CD3-zeta, CD28, CD27,4-1BB, and the like. In some instances, the CAR can comprise anycombination of CD3-zeta, CD28, 4-1BB, and the like.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the nucleic acidmolecule, by deriving the nucleic acid molecule from a vector known toinclude the same, or by isolating directly from cells and tissuescontaining the same, using standard techniques. Alternatively, thenucleic acid of interest can be produced synthetically, rather thancloned.

The present invention includes retroviral and lentiviral vectorconstructs expressing a CAR that can be directly transduced into a cell.

The present invention also includes an RNA construct that can bedirectly transfected into a cell. A method for generating mRNA for usein transfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”) (e.g., a 3′and/or 5′ UTR described herein), a 5′ cap (e.g., a 5′ cap describedherein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRESdescribed herein), the nucleic acid to be expressed, and a polyA tail,typically 50-2000 bases in length (SEQ ID NO:32). RNA so produced canefficiently transfect different kinds of cells. In one embodiment, thetemplate includes sequences for the CAR. In an embodiment, an RNA CARvector is transduced into a cell, e.g., a T cell or a NK cell, byelectroporation.

Antigen Binding Domain

In one aspect, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thus,examples of cell surface markers that may act as ligands for the antigenbinding domain in a CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one aspect, the CAR-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen binding domain thatspecifically binds a desired antigen into the CAR.

In one aspect, the portion of the CAR comprising the antigen bindingdomain comprises an antigen binding domain that targets a tumor antigen,e.g., a tumor antigen described herein.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, a T cell receptor (TCR), or a fragment there of,e.g., single chain TCR, and the like. In some instances, it isbeneficial for the antigen binding domain to be derived from the samespecies in which the CAR will ultimately be used in. For example, foruse in humans, it may be beneficial for the antigen binding domain ofthe CAR to comprise human or humanized residues for the antigen bindingdomain of an antibody or antibody fragment.

In one embodiment, an antigen binding domain against CD22 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Haso etal., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83-88 (2013);Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In one embodiment, an antigen binding domain against CS-1 is an antigenbinding portion, e.g., CDRs, of Elotuzumab (BMS), see e.g., Tai et al.,2008, Blood 112(4):1329-37; Tai et al., 2007, Blood. 110(5):1656-63.

In one embodiment, an antigen binding domain against CLL-1 is an antigenbinding portion, e.g., CDRs, of an antibody available from R&D,ebiosciences, Abcam, for example, PE-CLL1-hu Cat#353604 (BioLegend); andPE-CLL1 (CLEC12A) Cat#562566 (BD).

In one embodiment, an antigen binding domain against CD33 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Bross etal., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin,hP67.6), Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab,HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012)(AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and Pizzitola etal., Leukemia doi:10.1038/Lue.2014.62 (2014).

In one embodiment, an antigen binding domain against GD2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo etal., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440(1987), Cheung et al., J Clin Oncol 16(9):3053-3060 (1998),Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). Insome embodiments, an antigen binding domain against GD2 is an antigenbinding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18,hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g.,WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061,WO2013074916, and WO201385552. In some embodiments, an antigen bindingdomain against GD2 is an antigen binding portion of an antibodydescribed in US Publication No.: 20100150910 or PCT Publication No.: WO2011160119.

In one embodiment, an antigen binding domain against BCMA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,WO2012163805, WO200112812, and WO2003062401.

In one embodiment, an antigen binding domain against Tn antigen is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,U.S. Pat. No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010),and Stone et al., OncoImmunology 1(6):863-873(2012).

In one embodiment, an antigen binding domain against PSMA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Parkeret al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013)(scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F11) and single chainantibody fragments (scFv A5 and D7).

In one embodiment, an antigen binding domain against ROR1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hudeceket al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; andUS20130101607.

In one embodiment, an antigen binding domain against FLT3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,WO2011076922, U.S. Pat. No. 5,777,084, EP0754230, US20090297529, andseveral commercial catalog antibodies (R&D, ebiosciences, Abcam).

In one embodiment, an antigen binding domain against TAG72 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hombachet al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.

In one embodiment, an antigen binding domain against FAP is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5),US Pat. Publication No. 2009/0304718; sibrotuzumab (see e.g., Hofheinzet al., Oncology Research and Treatment 26(1), 2003); and Tran et al., JExp Med 210(6):1125-1135 (2013).

In one embodiment, an antigen binding domain against CD38 is an antigenbinding portion, e.g., CDRs, of daratumumab (see, e.g., Groen et al.,Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., U.S. Pat. No.8,263,746); or antibodies described in U.S. Pat. No. 8,362,211.

In one embodiment, an antigen binding domain against CD44v6 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Casucci et al., Blood 122(20):3461-3472 (2013).

In one embodiment, an antigen binding domain against CEA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).

In one embodiment, an antigen binding domain against EPCAM is an antigenbinding portion, e.g., CDRS, of an antibody selected from MT110,EpCAM-CD3 bispecific Ab (see, e.g.,clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1;and adecatumumab (MT201).

In one embodiment, an antigen binding domain against PRSS21 is anantigen binding portion, e.g., CDRs, of an antibody described in U.S.Pat. No. 8,080,650.

In one embodiment, an antigen binding domain against B7H3 is an antigenbinding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics).

In one embodiment, an antigen binding domain against KIT is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,915,391, US20120288506, and several commercial catalogantibodies.

In one embodiment, an antigen binding domain against IL-13Ra2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,WO2008/146911, WO2004087758, several commercial catalog antibodies, andWO2004087758.

In one embodiment, an antigen binding domain against CD30 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,090,843 B1, and EP0805871.

In one embodiment, an antigen binding domain against GD3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,253,263; U.S. Pat. No. 8,207,308; US 20120276046; EP1013761;WO2005035577; and U.S. Pat. No. 6,437,098.

In one embodiment, an antigen binding domain against CD171 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hong etal., J Immunother 37(2):93-104 (2014).

In one embodiment, an antigen binding domain against IL-11Ra is anantigen binding portion, e.g., CDRs, of an antibody available from Abcam(cat# ab55262) or Novus Biologicals (cat# EPR5446). In anotherembodiment, an antigen binding domain again IL-11Ra is a peptide, see,e.g., Huang et al., Cancer Res 72(1):271-281 (2012).

In one embodiment, an antigen binding domain against PSCA is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5);Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFvC5-II); and US Pat Publication No. 20090311181.

In one embodiment, an antigen binding domain against VEGFR2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).

In one embodiment, an antigen binding domain against LewisY is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab(scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10scFv).

In one embodiment, an antigen binding domain against CD24 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Maliaret al., Gastroenterology 143(5):1375-1384 (2012).

In one embodiment, an antigen binding domain against PDGFR-beta is anantigen binding portion, e.g., CDRs, of an antibody Abcam ab32570.

In one embodiment, an antigen binding domain against SSEA-4 is anantigen binding portion, e.g., CDRs, of antibody MC813 (Cell Signaling),or other commercially available antibodies.

In one embodiment, an antigen binding domain against CD20 is an antigenbinding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab,Ocrelizumab, Veltuzumab, or GA101.

In one embodiment, an antigen binding domain against Folate receptoralpha is an antigen binding portion, e.g., CDRs, of the antibodyIMGN853, or an antibody described in US20120009181; U.S. Pat. No.4,851,332, LK26: U.S. Pat. No. 5,952,484.

In one embodiment, an antigen binding domain against ERBB2 (Her2/neu) isan antigen binding portion, e.g., CDRs, of the antibody trastuzumab, orpertuzumab.

In one embodiment, an antigen binding domain against MUC1 is an antigenbinding portion, e.g., CDRs, of the antibody SAR566658.

In one embodiment, the antigen binding domain against EGFR is antigenbinding portion, e.g., CDRs, of the antibody cetuximab, panitumumab,zalutumumab, nimotuzumab, or matuzumab.

In one embodiment, an antigen binding domain against NCAM is an antigenbinding portion, e.g., CDRs, of the antibody clone 2-2B: MAB5324 (EMDMillipore)

In one embodiment, an antigen binding domain against Ephrin B2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Abengozar et al., Blood 119(19):4565-4576 (2012).

In one embodiment, an antigen binding domain against IGF-I receptor isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., U.S. Pat. No. 8,344,112 B2; EP2322550 A1; WO 2006/138315, orPCT/US2006/022995.

In one embodiment, an antigen binding domain against CAIX is an antigenbinding portion, e.g., CDRs, of the antibody clone 303123 (R&D Systems).

In one embodiment, an antigen binding domain against LMP2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,410,640, or US20050129701.

In one embodiment, an antigen binding domain against gp100 is an antigenbinding portion, e.g., CDRs, of the antibody HMB45, NKIbetaB, or anantibody described in WO2013165940, or US20130295007

In one embodiment, an antigen binding domain against tyrosinase is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,U.S. Pat. No. 5,843,674; or US19950504048.

In one embodiment, an antigen binding domain against EphA2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Yu etal., Mol Ther 22(1):102-111 (2014).

In one embodiment, an antigen binding domain against GD3 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 7,253,263; U.S. Pat. No. 8,207,308; US 20120276046; EP1013761A3; 20120276046; WO2005035577; or U.S. Pat. No. 6,437,098.

In one embodiment, an antigen binding domain against fucosyl GM1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,US20100297138; or WO2007/067992.

In one embodiment, an antigen binding domain against sLe is an antigenbinding portion, e.g., CDRs, of the antibody G193 (for lewis Y), seeScott A M et al, Cancer Res 60: 3254-61 (2000), also as described inNeeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement)177.10.

In one embodiment, an antigen binding domain against GM3 is an antigenbinding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7).

In one embodiment, an antigen binding domain against HMWMAA is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382)(mAb9.2.27); U.S. Pat. No. 6,528,481; WO2010033866; or US 20140004124.

In one embodiment, an antigen binding domain against o-acetyl-GD2 is anantigen binding portion, e.g., CDRs, of the antibody 8B6.

In one embodiment, an antigen binding domain against TEM1/CD248 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J ImmunolMethods 363(2):221-232 (2011).

In one embodiment, an antigen binding domain against CLDN6 is an antigenbinding portion, e.g., CDRs, of the antibody IMAB027 (GanymedPharmaceuticals), see e.g., clinicaltrial.gov/show/NCT02054351.

In one embodiment, an antigen binding domain against TSHR is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 8,603,466; U.S. Pat. No. 8,501,415; or U.S. Pat. No. 8,309,693.

In one embodiment, an antigen binding domain against GPRC5D is anantigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&DSystems); or LS-A4180 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD97 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., U.S.Pat. No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009);or an antibody from R&D: MAB3734.

In one embodiment, an antigen binding domain against ALK is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g.,Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).

In one embodiment, an antigen binding domain against polysialic acid isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., Nagae et al., J Biol Chem 288(47):33784-33796 (2013).

In one embodiment, an antigen binding domain against PLAC1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Ghods etal., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177.

In one embodiment, an antigen binding domain against GloboH is anantigen binding portion of the antibody VK9; or an antibody describedin, e.g., Kudryashov V et al, Glycoconj J. 15(3):243-9 (1998), Lou etal., Proc Natl Acad Sci USA 111(7):2482-2487 (2014); MBr1: Bremer E-G etal. J Biol Chem 259:14773-14777 (1984).

In one embodiment, an antigen binding domain against NY-BR-1 is anantigen binding portion, e.g., CDRs of an antibody described in, e.g.,Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

In one embodiment, an antigen binding domain against WT-1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Dao etal., Sci Transl Med 5(176):176ra33 (2013); or WO2012/135854.

In one embodiment, an antigen binding domain against MAGE-A1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).

In one embodiment, an antigen binding domain against sperm protein 17 isan antigen binding portion, e.g., CDRs, of an antibody described in,e.g., Song et al., Target Oncol 2013 Aug. 14 (PMID: 23943313); Song etal., Med Oncol 29(4):2923-2931 (2012).

In one embodiment, an antigen binding domain against Tie 2 is an antigenbinding portion, e.g., CDRs, of the antibody AB33 (Cell SignalingTechnology).

In one embodiment, an antigen binding domain against MAD-CT-2 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,PMID: 2450952; U.S. Pat. No. 7,635,753.

In one embodiment, an antigen binding domain against Fos-related antigen1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (NovusBiologicals).

In one embodiment, an antigen binding domain against MelanA/MART1 is anantigen binding portion, e.g., CDRs, of an antibody described in,EP2514766 A2; or U.S. Pat. No. 7,749,719.

In one embodiment, an antigen binding domain against sarcomatranslocation breakpoints is an antigen binding portion, e.g., CDRs, ofan antibody described in, e.g., Luo et al, EMBO Mol. Med. 4(6):453-461(2012).

In one embodiment, an antigen binding domain against TRP-2 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Wang etal, J Exp Med. 184(6):2207-16 (1996).

In one embodiment, an antigen binding domain against CYP1B1 is anantigen binding portion, e.g., CDRs, of an antibody described in, e.g.,Maecker et al, Blood 102 (9): 3287-3294 (2003).

In one embodiment, an antigen binding domain against RAGE-1 is anantigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMDMillipore).

In one embodiment, an antigen binding domain against human telomerasereverse transcriptase is an antigen binding portion, e.g., CDRs, of theantibody cat no: LS-B95-100 (Lifespan Biosciences)

In one embodiment, an antigen binding domain against intestinal carboxylesterase is an antigen binding portion, e.g., CDRs, of the antibody4F12: cat no: LS-B6190-50 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against mut hsp70-2 is anantigen binding portion, e.g., CDRs, of the antibody LifespanBiosciences: monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD79a is an antigenbinding portion, e.g., CDRs, of the antibody Anti-CD79a antibody[HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351available from Cell Signalling Technology; or antibodyHPA017748—Anti-CD79A antibody produced in rabbit, available from SigmaAldrich.

In one embodiment, an antigen binding domain against CD79b is an antigenbinding portion, e.g., CDRs, of the antibody polatuzumab vedotin,anti-CD79b described in Dornan et al., “Therapeutic potential of ananti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for thetreatment of non-Hodgkin lymphoma” Blood. 2009 Sep. 24; 114(13):2721-9.doi: 10.1182/blood-2009-02-205500. Epub 2009 Jul. 24, or the bispecificantibody Anti-CD79b/CD3 described in “4507 Pre-Clinical Characterizationof T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a PotentialTherapy for B Cell Malignancies” Abstracts of 56^(th) ASH Annual Meetingand Exposition, San Francisco, Calif. Dec. 6-9 2014.

In one embodiment, an antigen binding domain against CD72 is an antigenbinding portion, e.g., CDRs, of the antibody J3-109 described in Myers,and Uckun, “An anti-CD72 immunotoxin against therapy-refractoryB-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 June;18(1-2):119-22, or anti-CD72 (10D6.8.1, mIgG1) described in Polson etal., “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin'sLymphoma: Target and Linker-Drug Selection” Cancer Res Mar. 15, 2009 69;2358.

In one embodiment, an antigen binding domain against LAIR1 is an antigenbinding portion, e.g., CDRs, of the antibody ANT-301 LAIR1 antibody,available from ProSpec; or anti-human CD305 (LAIR1) Antibody, availablefrom BioLegend.

In one embodiment, an antigen binding domain against FCAR is an antigenbinding portion, e.g., CDRs, of the antibody CD89/FCARAntibody(Catalog#10414-H08H), available from Sino Biological Inc.

In one embodiment, an antigen binding domain against LILRA2 is anantigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonalantibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2antibody, Monoclonal (2D7), available from Lifespan Biosciences.

In one embodiment, an antigen binding domain against CD300LF is anantigen binding portion, e.g., CDRs, of the antibody MouseAnti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available fromBioLegend, or Rat Anti-CMRF35-like molecule 1 antibody,Monoclonal[234903], available from R&D Systems.

In one embodiment, an antigen binding domain against CLEC12A is anantigen binding portion, e.g., CDRs, of the antibody Bispecific T cellEngager (BiTE) scFv-antibody and ADC described in Noordhuis et al.,“Targeting of CLEC12A In Acute Myeloid Leukemia byAntibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody” 53^(rd)ASH Annual Meeting and Exposition, Dec. 10-13, 2011, and MCLA-117(Merus).

In one embodiment, an antigen binding domain against BST2 (also calledCD317) is an antigen binding portion, e.g., CDRs, of the antibody MouseAnti-CD317 antibody, Monoclonal[3H4], available from Antibodies-Onlineor Mouse Anti-CD317 antibody, Monoclonal[696739], available from R&DSystems.

In one embodiment, an antigen binding domain against EMR2 (also calledCD312) is an antigen binding portion, e.g., CDRs, of the antibody MouseAnti-CD312 antibody, Monoclonal[LS-B8033] available from LifespanBiosciences, or Mouse Anti-CD312 antibody, Monoclonal[494025] availablefrom R&D Systems.

In one embodiment, an antigen binding domain against LY75 is an antigenbinding portion, e.g., CDRs, of the antibody Mouse Anti-Lymphocyteantigen 75 antibody, Monoclonal[HD30] available from EMD Millipore orMouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[A15797] availablefrom Life Technologies.

In one embodiment, an antigen binding domain against GPC3 is an antigenbinding portion, e.g., CDRs, of the antibody hGC33 described in NakanoK, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican3 antibody by CDR grafting and stability optimization. Anticancer Drugs.2010 November; 21(10):907-916, or MDX-1414, HN3, or YP7, all three ofwhich are described in Feng et al., “Glypican-3 antibodies: a newtherapeutic target for liver cancer.” FEBS Lett. 2014 Jan. 21;588(2):377-82.

In one embodiment, an antigen binding domain against FCRL5 is an antigenbinding portion, e.g., CDRs, of the anti-FcRL5 antibody described inElkins et al., “FcRL5 as a target of antibody-drug conjugates for thetreatment of multiple myeloma” Mol Cancer Ther. 2012 October;11(10):2222-32.

In one embodiment, an antigen binding domain against IGLL1 is an antigenbinding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulinlambda-like polypeptide 1 antibody, Monoclonal[AT1G4] available fromLifespan Biosciences, Mouse Anti-Immunoglobulin lambda-like polypeptide1 antibody, Monoclonal[HSL11] available from BioLegend.

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted above.

In another aspect, the antigen binding domain comprises a humanizedantibody or an antibody fragment. In some aspects, a non-human antibodyis humanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman or fragment thereof. In one aspect, the antigen binding domain ishumanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886,International Publication No. WO 9317105, Tan et al., J. Immunol.,169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000),Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem.,272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904(1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995),Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene,150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73(1994), each of which is incorporated herein in its entirety byreference. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, for example improve, antigen binding. These frameworksubstitutions are identified by methods well-known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized antibodies and antibody fragments, substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a nonhuman species. Humanized antibodies areoften human antibodies in which some CDR residues and possibly someframework (FR) residues are substituted by residues from analogous sitesin rodent antibodies. Humanization of antibodies and antibody fragmentscan also be achieved by veneering or resurfacing (EP 592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al.,PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332),the contents of which are incorporated herein by reference herein intheir entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (see, e.g., Nicholson et al. Mol. Immun 34 (16-17): 1157-1165(1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety). In someembodiments, the framework region, e.g., all four framework regions, ofthe heavy chain variable region are derived from a VH4_4-59 germlinesequence. In one embodiment, the framework region can comprise, one,two, three, four or five modifications, e.g., substitutions, e.g., fromthe amino acid at the corresponding murine sequence. In one embodiment,the framework region, e.g., all four framework regions of the lightchain variable region are derived from a VK3_1.25 germline sequence. Inone embodiment, the framework region can comprise, one, two, three, fouror five modifications, e.g., substitutions, e.g., from the amino acid atthe corresponding murine sequence.

In some aspects, the portion of a CAR composition of the invention thatcomprises an antibody fragment is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies and antibody fragments are prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, e.g., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody or antibody fragment characteristic, such asincreased affinity for the target antigen, is achieved. In general, theCDR residues are directly and most substantially involved in influencingantigen binding.

A humanized antibody or antibody fragment may retain a similar antigenicspecificity as the original antibody, e.g., in the present invention,the ability to bind human a cancer associated antigen as describedherein. In some embodiments, a humanized antibody or antibody fragmentmay have improved affinity and/or specificity of binding to human acancer associated antigen as described herein.

In one aspect, the antigen binding domain of the invention ischaracterized by particular functional features or properties of anantibody or antibody fragment. For example, in one aspect, the portionof a CAR composition of the invention that comprises an antigen bindingdomain specifically binds a tumor antigen as described herein.

In one aspect, the anti-cancer associated antigen as described hereinbinding domain is a fragment, e.g., a single chain variable fragment(scFv). In one aspect, the anti-cancer associated antigen as describedherein binding domain is a Fv, a Fab, a (Fab′)2, or a bifunctional (e.g.bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J.Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragmentsthereof of the invention binds a cancer associated antigen as describedherein protein with wild-type or enhanced affinity.

In some instances, scFvs can be prepared according to method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids) intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, is incorporated hereinby reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n, where n is a positive integer equal to orgreater than 1 (SEQ ID NO:22). In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO:29) or (Gly₄Ser)₃(SEQ ID NO:30). Variation in thelinker length may retain or enhance activity, giving rise to superiorefficacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor(“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).Methods to make such TCRs are known in the art. See, e.g., Willemsen R Aet al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012)(references are incorporated herein by its entirety). For example, scTCRcan be engineered that contains the Vα and Vβ genes from a T cell clonelinked by a linker (e.g., a flexible peptide). This approach is veryuseful to cancer associated target that itself is intracellular,however, a fragment of such antigen (peptide) is presented on thesurface of the cancer cells by MHC.

Bispecific CARs

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Protocols forgenerating bispecific or heterodimeric antibody molecules are known inthe art; including but not limited to, for example, the “knob in a hole”approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostaticsteering Fc pairing as described in, e.g., WO 09/089004, WO 06/106905and WO 2010/129304; Strand Exchange Engineered Domains (SEED)heterodimer formation as described in, e.g., WO 07/110205; Fab armexchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO2013/060867; double antibody conjugate, e.g., by antibody cross-linkingto generate a bi-specific structure using a heterobifunctional reagenthaving an amine-reactive group and a sulfhydryl reactive group asdescribed in, e.g., U.S. Pat. No. 4,433,059; bispecific antibodydeterminants generated by recombining half antibodies (heavy-light chainpairs or Fabs) from different antibodies through cycle of reduction andoxidation of disulfide bonds between the two heavy chains, as describedin, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., threeFab′ fragments cross-linked through sulfhydryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743; biosynthetic bindingproteins, e.g., pair of scFvs cross-linked through C-terminal tailspreferably through disulfide or amine-reactive chemical cross-linking,as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies,e.g., Fab fragments with different binding specificities dimerizedthrough leucine zippers (e.g., c-fos and c-jun) that have replaced theconstant domain, as described in, e.g., U.S. Pat. No. 5,582,996;bispecific and oligospecific mono- and oligovalent receptors, e.g.,VH-CH1 regions of two antibodies (two Fab fragments) linked through apolypeptide spacer between the CH1 region of one antibody and the VHregion of the other antibody typically with associated light chains, asdescribed in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibodyconjugates, e.g., crosslinking of antibodies or Fab fragments through adouble stranded piece of DNA, as described in, e.g., U.S. Pat. No.5,635,602; bispecific fusion proteins, e.g., an expression constructcontaining two scFvs with a hydrophilic helical peptide linker betweenthem and a full constant region, as described in, e.g., U.S. Pat. No.5,637,481; multivalent and multispecific binding proteins, e.g., dimerof polypeptides having first domain with binding region of Ig heavychain variable region, and second domain with binding region of Ig lightchain variable region, generally termed diabodies (higher orderstructures are also encompassed creating for bispecific, trispecific, ortetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a shortpeptide linker (e.g., 5 or 10 amino acids) or no linker at all in eitherorientation, which can form dimers to form bispecific diabodies; trimersand tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptidelinkages with crosslinkable groups at the C-terminus further associatedwith VL domains to form a series of FVs (or scFvs), as described in,e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptideswith both a VH and a VL domain linked through a peptide linker arecombined into multivalent structures through non-covalent or chemicalcrosslinking to form, e.g., homobivalent, heterobivalent, trivalent, andtetravalent structures using both scFV or diabody type format, asdescribed in, e.g., U.S. Pat. No. 5,869,620. Additional exemplarymultispecific and bispecific molecules and methods of making the sameare found, for example, in U.S. Pat. No. 5,910,573, U.S. Pat. No.5,932,448, U.S. Pat. No. 5,959,083, U.S. Pat. No. 5,989,830, U.S. Pat.No. 6,005,079, U.S. Pat. No. 6,239,259, U.S. Pat. No. 6,294,353, U.S.Pat. No. 6,333,396, U.S. Pat. No. 6,476,198, U.S. Pat. No. 6,511,663,U.S. Pat. No. 6,670,453, U.S. Pat. No. 6,743,896, U.S. Pat. No.6,809,185, U.S. Pat. No. 6,833,441, U.S. Pat. No. 7,129,330, U.S. Pat.No. 7,183,076, U.S. Pat. No. 7,521,056, U.S. Pat. No. 7,527,787, U.S.Pat. No. 7,534,866, U.S. Pat. No. 7,612,181, US2002004587A1,US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1,US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1,US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1,US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1,US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1,US2007004909A1, US2007087381A1, US2007128150A1, US2007141049A1,US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1,US2008152645A1, US2008171855A1, US2008241884A1, US2008254512A1,US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1,US2009162359A1, US2009162360A1, US2009175851A1, US2009175867A1,US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1,EP346087A2, W00006605A2, WO02072635A2, WO04081051A1, WO06020258A2,WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1,WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1,WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1. The contents of theabove-referenced applications are incorporated herein by reference intheir entireties.

Within each antibody or antibody fragment (e.g., scFv) of a bispecificantibody molecule, the VH can be upstream or downstream of the VL. Insome embodiments, the upstream antibody or antibody fragment (e.g.,scFv) is arranged with its VH (VH₁) upstream of its VL (VL₁) and thedownstream antibody or antibody fragment (e.g., scFv) is arranged withits VL (VL₂) upstream of its VH (VH₂), such that the overall bispecificantibody molecule has the arrangement VH₁-VL₁-VL₂-VH₂. In otherembodiments, the upstream antibody or antibody fragment (e.g., scFv) isarranged with its VL (VL₁) upstream of its VH (VH₁) and the downstreamantibody or antibody fragment (e.g., scFv) is arranged with its VH (VH₂)upstream of its VL (VL₂), such that the overall bispecific antibodymolecule has the arrangement VL₁-VH₁-VH₂-VL₂. Optionally, a linker isdisposed between the two antibodies or antibody fragments (e.g., scFvs),e.g., between VL₁ and VL₂ if the construct is arranged asVH₁-VL₁-VL₂-VH₂, or between VH₁ and VH₂ if the construct is arranged asVL₁-VH₁-VH₂-VL₂. The linker may be a linker as described herein, e.g., a(Gly₄-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQID NO: 78). In general, the linker between the two scFvs should be longenough to avoid mispairing between the domains of the two scFvs.Optionally, a linker is disposed between the VL and VH of the firstscFv. Optionally, a linker is disposed between the VL and VH of thesecond scFv. In constructs that have multiple linkers, any two or moreof the linkers can be the same or different. Accordingly, in someembodiments, a bispecific CAR comprises VLs, VHs, and optionally one ormore linkers in an arrangement as described herein.

Stability and Mutations

The stability of an antigen binding domain to a cancer associatedantigen as described herein, e.g., scFv molecules (e.g., soluble scFv),can be evaluated in reference to the biophysical properties (e.g.,thermal stability) of a conventional control scFv molecule or a fulllength antibody. In one embodiment, the humanized scFv has a thermalstability that is greater than about 0.1, about 0.25, about 0.5, about0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5,about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6,about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5,about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees,about 14 degrees, or about 15 degrees Celsius than a control bindingmolecule (e.g. a conventional scFv molecule) in the described assays.

The improved thermal stability of the antigen binding domain to a cancerassociated antigen described herein, e.g., scFv is subsequentlyconferred to the entire CAR construct, leading to improved therapeuticproperties of the CAR construct. The thermal stability of the antigenbinding domain of -a cancer associated antigen described herein, e.g.,scFv, can be improved by at least about 2° C. or 3° C. as compared to aconventional antibody. In one embodiment, the antigen binding domain of-a cancer associated antigen described herein, e.g., scFv, has a 1° C.improved thermal stability as compared to a conventional antibody. Inanother embodiment, the antigen binding domain of a cancer associatedantigen described herein, e.g., scFv, has a 2° C. improved thermalstability as compared to a conventional antibody. In another embodiment,the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C. improvedthermal stability as compared to a conventional antibody. Comparisonscan be made, for example, between the scFv molecules disclosed hereinand scFv molecules or Fab fragments of an antibody from which the scFvVH and VL were derived. Thermal stability can be measured using methodsknown in the art. For example, in one embodiment, Tm can be measured.Methods for measuring Tm and other methods of determining proteinstability are described in more detail below.

Mutations in scFv (arising through humanization or direct mutagenesis ofthe soluble scFv) can alter the stability of the scFv and improve theoverall stability of the scFv and the CAR construct. Stability of thehumanized scFv is compared against the murine scFv using measurementssuch as Tm, temperature denaturation and temperature aggregation.

The binding capacity of the mutant scFvs can be determined using assaysknow in the art and described herein.

In one embodiment, the antigen binding domain of -a cancer associatedantigen described herein, e.g., scFv, comprises at least one mutationarising from the humanization process such that the mutated scFv confersimproved stability to the CAR construct. In another embodiment, theantigen binding domain of -a cancer associated antigen described herein,e.g., scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutationsarising from the humanization process such that the mutated scFv confersimproved stability to the CAR construct.

Methods of Evaluating Protein Stability

The stability of an antigen binding domain may be assessed using, e.g.,the methods described below. Such methods allow for the determination ofmultiple thermal unfolding transitions where the least stable domaineither unfolds first or limits the overall stability threshold of amultidomain unit that unfolds cooperatively (e.g., a multidomain proteinwhich exhibits a single unfolding transition). The least stable domaincan be identified in a number of additional ways. Mutagenesis can beperformed to probe which domain limits the overall stability.Additionally, protease resistance of a multidomain protein can beperformed under conditions where the least stable domain is known to beintrinsically unfolded via DSC or other spectroscopic methods (Fontana,et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol.393: 672-692). Once the least stable domain is identified, the sequenceencoding this domain (or a portion thereof) may be employed as a testsequence in the methods.

Thermal Stability

The thermal stability of the compositions may be analyzed using a numberof non-limiting biophysical or biochemical techniques known in the art.In certain embodiments, thermal stability is evaluated by analyticalspectroscopy.

An exemplary analytical spectroscopy method is Differential Scanningcalorimetry (DSC). DSC employs a calorimeter which is sensitive to theheat absorbances that accompany the unfolding of most proteins orprotein domains (see, e.g. Sanchez-Ruiz, et al., Biochemistry, 27:1648-52, 1988). To determine the thermal stability of a protein, asample of the protein is inserted into the calorimeter and thetemperature is raised until the Fab or scFv unfolds. The temperature atwhich the protein unfolds is indicative of overall protein stability.

Another exemplary analytical spectroscopy method is Circular Dichroism(CD) spectroscopy. CD spectrometry measures the optical activity of acomposition as a function of increasing temperature. Circular dichroism(CD) spectroscopy measures differences in the absorption of left-handedpolarized light versus right-handed polarized light which arise due tostructural asymmetry. A disordered or unfolded structure results in a CDspectrum very different from that of an ordered or folded structure. TheCD spectrum reflects the sensitivity of the proteins to the denaturingeffects of increasing temperature and is therefore indicative of aprotein's thermal stability (see van Mierlo and Steemsma, J.Biotechnol., 79(3):281-98, 2000).

Another exemplary analytical spectroscopy method for measuring thermalstability is Fluorescence Emission Spectroscopy (see van Mierlo andSteemsma, supra). Yet another exemplary analytical spectroscopy methodfor measuring thermal stability is Nuclear Magnetic Resonance (NMR)spectroscopy (see, e.g. van Mierlo and Steemsma, supra).

The thermal stability of a composition can be measured biochemically. Anexemplary biochemical method for assessing thermal stability is athermal challenge assay. In a “thermal challenge assay”, a compositionis subjected to a range of elevated temperatures for a set period oftime. For example, in one embodiment, test scFv molecules or moleculescomprising scFv molecules are subject to a range of increasingtemperatures, e.g., for 1-1.5 hours. The activity of the protein is thenassayed by a relevant biochemical assay. For example, if the protein isa binding protein (e.g. an scFv or scFv-containing polypeptide) thebinding activity of the binding protein may be determined by afunctional or quantitative ELISA.

Such an assay may be done in a high-throughput format and thosedisclosed in the Examples using E. coli and high throughput screening. Alibrary of antigen binding domains, e.g., that includes an antigenbinding domain to -a cancer associated antigen described herein, e.g.,scFv variants, may be created using methods known in the art. Antigenbinding domain, e.g., to -a cancer associated antigen described herein,e.g., scFv, expression may be induced and the antigen binding domain,e.g., to -a cancer associated antigen described herein, e.g., scFv, maybe subjected to thermal challenge. The challenged test samples may beassayed for binding and those antigen binding domains to -a cancerassociated antigen described herein, e.g., scFvs, which are stable maybe scaled up and further characterized.

Thermal stability is evaluated by measuring the melting temperature (Tm)of a composition using any of the above techniques (e.g. analyticalspectroscopy techniques). The melting temperature is the temperature atthe midpoint of a thermal transition curve wherein 50% of molecules of acomposition are in a folded state (See e.g., Dimasi et al. (2009) J. MolBiol. 393: 672-692). In one embodiment, Tm values for an antigen bindingdomain to -a cancer associated antigen described herein, e.g., scFv, areabout 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C.,48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C.,57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C.,66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C.,75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C.,84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C.,93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C. In oneembodiment, Tm values for an IgG is about 40° C., 41° C., 42° C., 43°C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52°C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61°C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70°C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79°C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88°C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97°C., 98° C., 99° C., 100° C. In one embodiment, Tm values for anmultivalent antibody is about 40° C., 41° C., 42° C., 43° C., 44° C.,45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C.,54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C.,63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C.,72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C.,81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C.,90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C.,99° C., 100° C.

Thermal stability is also evaluated by measuring the specific heat orheat capacity (Cp) of a composition using an analytical calorimetrictechnique (e.g. DSC). The specific heat of a composition is the energy(e.g. in kcal/mol) is required to rise by 1° C., the temperature of 1mol of water. As large Cp is a hallmark of a denatured or inactiveprotein composition. The change in heat capacity (ΔCp) of a compositionis measured by determining the specific heat of a composition before andafter its thermal transition. Thermal stability may also be evaluated bymeasuring or determining other parameters of thermodynamic stabilityincluding Gibbs free energy of unfolding (ΔG), enthalpy of unfolding(ΔH), or entropy of unfolding (ΔS). One or more of the above biochemicalassays (e.g. a thermal challenge assay) are used to determine thetemperature (i.e. the T_(C) value) at which 50% of the compositionretains its activity (e.g. binding activity).

In addition, mutations to the antigen binding domain of a cancerassociated antigen described herein, e.g., scFv, can be made to alterthe thermal stability of the antigen binding domain of a cancerassociated antigen described herein, e.g., scFv, as compared with theunmutated antigen binding domain of a cancer associated antigendescribed herein, e.g., scFv. When the humanized antigen binding domainof a cancer associated antigen described herein, e.g., scFv, isincorporated into a CAR construct, the antigen binding domain of thecancer associated antigen described herein, e.g., humanized scFv,confers thermal stability to the overall CARs of the present invention.In one embodiment, the antigen binding domain to a cancer associatedantigen described herein, e.g., scFv, comprises a single mutation thatconfers thermal stability to the antigen binding domain of the cancerassociated antigen described herein, e.g., scFv. In another embodiment,the antigen binding domain to a cancer associated antigen describedherein, e.g., scFv, comprises multiple mutations that confer thermalstability to the antigen binding domain to the cancer associated antigendescribed herein, e.g., scFv. In one embodiment, the multiple mutationsin the antigen binding domain to a cancer associated antigen describedherein, e.g., scFv, have an additive effect on thermal stability of theantigen binding domain to the cancer associated antigen described hereinbinding domain, e.g., scFv.

b) % Aggregation

The stability of a composition can be determined by measuring itspropensity to aggregate. Aggregation can be measured by a number ofnon-limiting biochemical or biophysical techniques. For example, theaggregation of a composition may be evaluated using chromatography, e.g.Size-Exclusion Chromatography (SEC). SEC separates molecules on thebasis of size. A column is filled with semi-solid beads of a polymericgel that will admit ions and small molecules into their interior but notlarge ones. When a protein composition is applied to the top of thecolumn, the compact folded proteins (i.e. non-aggregated proteins) aredistributed through a larger volume of solvent than is available to thelarge protein aggregates. Consequently, the large aggregates move morerapidly through the column, and in this way the mixture can be separatedor fractionated into its components. Each fraction can be separatelyquantified (e.g. by light scattering) as it elutes from the gel.Accordingly, the % aggregation of a composition can be determined bycomparing the concentration of a fraction with the total concentrationof protein applied to the gel. Stable compositions elute from the columnas essentially a single fraction and appear as essentially a single peakin the elution profile or chromatogram.

c) Binding Affinity

The stability of a composition can be assessed by determining its targetbinding affinity. A wide variety of methods for determining bindingaffinity are known in the art. An exemplary method for determiningbinding affinity employs surface plasmon resonance. Surface plasmonresonance is an optical phenomenon that allows for the analysis ofreal-time biospecific interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway,N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627;Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson,B., et al. (1991) Anal. Biochem. 198:268-277.

In one aspect, the antigen binding domain of the CAR comprises an aminoacid sequence that is homologous to an antigen binding domain amino acidsequence described herein, and the antigen binding domain retains thedesired functional properties of the antigen binding domain describedherein.

In one specific aspect, the CAR composition of the invention comprisesan antibody fragment. In a further aspect, the antibody fragmentcomprises an scFv.

In various aspects, the antigen binding domain of the CAR is engineeredby modifying one or more amino acids within one or both variable regions(e.g., VH and/or VL), for example within one or more CDR regions and/orwithin one or more framework regions. In one specific aspect, the CARcomposition of the invention comprises an antibody fragment. In afurther aspect, the antibody fragment comprises an scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, e.g., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over aspecified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, (1988)Comput. Appl. Biosci. 4:11-17) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the VH or VL ofan antigen binding domain to -a cancer associated antigen describedherein, e.g., scFv, comprised in the CAR can be modified to retain atleast about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identity of the starting VH or VL framework region ofthe antigen binding domain to the cancer associated antigen describedherein, e.g., scFv. The present invention contemplates modifications ofthe entire CAR construct, e.g., modifications in one or more amino acidsequences of the various domains of the CAR construct in order togenerate functionally equivalent molecules. The CAR construct can bemodified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting CARconstruct.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR e.g., inone embodiment, the transmembrane domain may be from the same proteinthat the signaling domain, costimulatory domain or the hinge domain isderived from. In another aspect, the transmembrane domain is not derivedfrom the same protein that any other domain of the CAR is derived from.In some instances, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membraneproteins, e.g., to minimize interactions with other members of thereceptor complex. In one aspect, the transmembrane domain is capable ofhomodimerization with another CAR on the cell surface of aCAR-expressing cell. In a different aspect, the amino acid sequence ofthe transmembrane domain may be modified or substituted so as tominimize interactions with the binding domains of the native bindingpartner present in the same CAR-expressing cell.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someembodiments, a transmembrane domain may include at least thetransmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a,CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2Rgamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b,ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108),SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR,PAG/Cbp, NKG2D, NKG2C.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge(e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linkerdescribed herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment,the hinge or spacer comprises (e.g., consists of) the amino acidsequence of SEQ ID NO:4. In one aspect, the transmembrane domaincomprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 12.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGKM (SEQ IDNO:6). In some embodiments, the hinge or spacer comprises a hingeencoded by a nucleotide sequence ofGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:7).

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYV TDH (SEQ IDNO:8). In some embodiments, the hinge or spacer comprises a hingeencoded by a nucleotide sequence ofAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO:9).

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:10).

In some embodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:11).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR includes an intracellularsignaling domain. An intracellular signaling domain is generallyresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been introduced. Theterm “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of CD3 zeta, commonFcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma,CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In oneembodiment, a CAR of the invention comprises an intracellular signalingdomain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs.

The intracellular signalling domain of the CAR can comprise the CD3-zetasignaling domain by itself or it can be combined with any other desiredintracellular signaling domain(s) useful in the context of a CAR of theinvention. For example, the intracellular signaling domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Forexample, CD27 costimulation has been demonstrated to enhance expansion,effector function, and survival of human CART cells in vitro andaugments human T cell persistence and antitumor activity in vivo (Songet al. Blood. 2012; 119(3):696-706). Further examples of suchcostimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55),PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, and CD19a.

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequence. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 14. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 18.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQID NO:16). In one aspect, the signalling domain of CD27 is encoded by anucleic acid sequence ofAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTT CGCAGCCTATCGCTCC(SEQ ID NO:17).

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target or a different target(e.g., a target other than a cancer associated antigen described hereinor a different cancer associated antigen described herein). In oneembodiment, the second CAR includes an antigen binding domain to atarget expressed the same cancer cell type as the cancer associatedantigen. In one embodiment, the CAR-expressing cell comprises a firstCAR that targets a first antigen and includes an intracellular signalingdomain having a costimulatory signaling domain but not a primarysignaling domain, and a second CAR that targets a second, different,antigen and includes an intracellular signaling domain having a primarysignaling domain but not a costimulatory signaling domain. While notwishing to be bound by theory, placement of a costimulatory signalingdomain, e.g., 4-1BB, CD28, CD27 or OX-40, onto the first CAR, and theprimary signaling domain, e.g., CD3 zeta, on the second CAR can limitthe CAR activity to cells where both targets are expressed. In oneembodiment, the CAR expressing cell comprises a first cancer associatedantigen CAR that includes an antigen binding domain that binds a targetantigen described herein, a transmembrane domain and a costimulatorydomain and a second CAR that targets a different target antigen (e.g.,an antigen expressed on that same cancer cell type as the first targetantigen) and includes an antigen binding domain, a transmembrane domainand a primary signaling domain. In another embodiment, the CARexpressing cell comprises a first CAR that includes an antigen bindingdomain that binds a target antigen described herein, a transmembranedomain and a primary signaling domain and a second CAR that targets anantigen other than the first target antigen (e.g., an antigen expressedon the same cancer cell type as the first target antigen) and includesan antigen binding domain to the antigen, a transmembrane domain and acostimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises an XCAR describedherein and an inhibitory CAR. In one embodiment, the inhibitory CARcomprises an antigen binding domain that binds an antigen found onnormal cells but not cancer cells, e.g., normal cells that also expressCLL. In one embodiment, the inhibitory CAR comprises the antigen bindingdomain, a transmembrane domain and an intracellular domain of aninhibitory molecule. For example, the intracellular domain of theinhibitory CAR can be an intracellular domain of PD1, PD-L1, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta.

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, the antigen binding domain comprises a singledomain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, lamprey, fish,shark, goat, rabbit, and bovine. This term also includes naturallyoccurring single domain antibody molecules from species other thanCamelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

It has also been discovered, that cells having a plurality of chimericmembrane embedded receptors comprising an antigen binding domain thatinteractions between the antigen binding domain of the receptors can beundesirable, e.g., because it inhibits the ability of one or more of theantigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are cells having a first and a second non-naturallyoccurring chimeric membrane embedded receptor comprising antigen bindingdomains that minimize such interactions. Also disclosed herein arenucleic acids encoding a first and a second non-naturally occurringchimeric membrane embedded receptor comprising antigen binding domainsthat minimize such interactions, as well as methods of making and usingsuch cells and nucleic acids. In an embodiment the antigen bindingdomain of one of said first said second non-naturally occurring chimericmembrane embedded receptor, comprises an scFv, and the other comprises asingle VH domain, e.g., a camelid, shark, or lamprey single VH domain,or a single VH domain derived from a human or mouse sequence.

In some embodiments, the claimed invention comprises a first and secondCAR, wherein the antigen binding domain of one of said first CAR saidsecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofsaid first CAR said second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof said first CAR said second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said first CARsaid second CAR comprises an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence. In some embodiments,the antigen binding domain of one of said first CAR said second CARcomprises an scFv, and the other comprises a nanobody. In someembodiments, the antigen binding domain of one of said first CAR saidsecond CAR comprises an scFv, and the other comprises a camelid VHHdomain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of said first CAR to its cognate antigen isnot substantially reduced by the presence of said second CAR. In someembodiments, binding of the antigen binding domain of said first CAR toits cognate antigen in the presence of said second CAR is 85%, 90%, 95%,96%, 97%, 98% or 99% of binding of the antigen binding domain of saidfirst CAR to its cognate antigen in the absence of said second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of said first CAR said second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of said first CAR said secondCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% lessthan if both were scFv antigen binding domains.

In another aspect, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity of aCAR-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,PD1, can, in some embodiments, decrease the ability of a CAR-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4and TGFR beta. In one embodiment, the agent which inhibits an inhibitorymolecule, e.g., is a molecule described herein, e.g., an agent thatcomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least aportion of an extracellular domain of any of these), and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27 or CD28,e.g., as described herein) and/or a primary signaling domain (e.g., aCD3 zeta signaling domain described herein). In one embodiment, theagent comprises a first polypeptide of PD1 or a fragment thereof (e.g.,at least a portion of an extracellular domain of PD1), and a secondpolypeptide of an intracellular signaling domain described herein (e.g.,a CD28 signaling domain described herein and/or a CD3 zeta signalingdomain described herein). PD1 is an inhibitory member of the CD28 familyof receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 isexpressed on activated B cells, T cells and myeloid cells (Agata et al.1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 havebeen shown to downregulate T cell activation upon binding to PD1(Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 NatImmunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 isabundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank etal. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 ClinCancer Res 10:5094) Immune suppression can be reversed by inhibiting thelocal interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1), fused to atransmembrane domain and intracellular signaling domains such as 41BBand CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment,the PD1 CAR, when used in combinations with a XCAR described herein,improves the persistence of the T cell. In one embodiment, the CAR is aPD1 CAR comprising the extracellular domain of PD1 indicated asunderlined in SEQ ID NO: 26. In one embodiment, the PD1 CAR comprisesthe amino acid sequence of SEQ ID NO:26.

(SEQ ID NO: 26) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeshaelryterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the PD1 CAR comprises the amino acid sequenceprovided below (SEQ ID NO:39).

(SEQ ID NO: 39) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfylnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeshaelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the agent comprises a nucleic acid sequence encodingthe PD1 CAR, e.g., the PD1 CAR described herein. In one embodiment, thenucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECDunderlined below in SEQ ID NO: 27

(SEQ ID NO: 27) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcaggagtgactgagggcgataatgcgaccacacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtaccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatagggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagatctgtacattacaagcagccatcatgaggcccgtgcaaaccacccaggaggaggacggagctcctgccggaccccgaagaggaagaaggaggagcgagctgcgcgtgaagactcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcaca tgcaggcccttccccctcgc.

In another aspect, the present invention provides a population ofCAR-expressing cells, e.g., CART cells. In some embodiments, thepopulation of CAR-expressing cells comprises a mixture of cellsexpressing different CARs. For example, in one embodiment, thepopulation of CART cells can include a first cell expressing a CARhaving an antigen binding domain to a cancer associated antigendescribed herein, and a second cell expressing a CAR having a differentantigen binding domain, e.g., an antigen binding domain to a different acancer associated antigen described herein, e.g., an antigen bindingdomain to a cancer associated antigen described herein that differs fromthe cancer associated antigen bound by the antigen binding domain of theCAR expressed by the first cell. As another example, the population ofCAR-expressing cells can include a first cell expressing a CAR thatincludes an antigen binding domain to a cancer associated antigendescribed herein, and a second cell expressing a CAR that includes anantigen binding domain to a target other than a cancer associatedantigen as described herein. In one embodiment, the population ofCAR-expressing cells includes, e.g., a first cell expressing a CAR thatincludes a primary intracellular signaling domain, and a second cellexpressing a CAR that includes a secondary signaling domain.

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a CAR having anantigen binding domain to a cancer associated antigen described herein,and a second cell expressing another agent, e.g., an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Inhibitory molecules, e.g., PD-1, can, in some embodiments,decrease the ability of a CAR-expressing cell to mount an immuneeffector response. Examples of inhibitory molecules include PD-1, PD-L1,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment,the agent which inhibits an inhibitory molecule, e.g., is a moleculedescribed herein, e.g., an agent that comprises a first polypeptide,e.g., an inhibitory molecule, associated with a second polypeptide thatprovides a positive signal to the cell, e.g., an intracellular signalingdomain described herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD-1, PD-L1, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any ofthese, and a second polypeptide which is an intracellular signalingdomain described herein (e.g., comprising a costimulatory domain (e.g.,41BB, CD27, OX40 or CD28, e.g., as described herein) and/or a primarysignaling domain (e.g., a CD3 zeta signaling domain described herein).In one embodiment, the agent comprises a first polypeptide of PD-1 or afragment thereof, and a second polypeptide of an intracellular signalingdomain described herein (e.g., a CD28 signaling domain described hereinand/or a CD3 zeta signaling domain described herein).

In one aspect, the present invention provides methods comprisingadministering a population of CAR-expressing cells, e.g., CART cells,e.g., a mixture of cells expressing different CARs, in combination withanother agent, e.g., a kinase inhibitor, such as a kinase inhibitordescribed herein. In another aspect, the present invention providesmethods comprising administering a population of cells wherein at leastone cell in the population expresses a CAR having an antigen bindingdomain of a cancer associated antigen described herein, and a secondcell expressing another agent, e.g., an agent which enhances theactivity of a CAR-expressing cell, in combination with another agent,e.g., a kinase inhibitor, such as a kinase inhibitor described herein.

Regulatable Chimeric Antigen Receptors

In some embodiments, a regulatable CAR (RCAR) where the CAR activity canbe controlled is desirable to optimize the safety and efficacy of a CARtherapy. There are many ways CAR activities can be regulated. Forexample, inducible apoptosis using, e.g., a caspase fused to adimerization domain (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3;365(18):1673-1683), can be used as a safety switch in the CAR therapy ofthe instant invention. In an aspect, a RCAR comprises a set ofpolypeptides, typically two in the simplest embodiments, in which thecomponents of a standard CAR described herein, e.g., an antigen bindingdomain and an intracellular signaling domain, are partitioned onseparate polypeptides or members. In some embodiments, the set ofpolypeptides include a dimerization switch that, upon the presence of adimerization molecule, can couple the polypeptides to one another, e.g.,can couple an antigen binding domain to an intracellular signalingdomain.

In an aspect, an RCAR comprises two polypeptides or members: 1) anintracellular signaling member comprising an intracellular signalingdomain, e.g., a primary intracellular signaling domain described herein,and a first switch domain; 2) an antigen binding member comprising anantigen binding domain, e.g., that targets a tumor antigen describedherein, as described herein and a second switch domain Optionally, theRCAR comprises a transmembrane domain described herein. In anembodiment, a transmembrane domain can be disposed on the intracellularsignaling member, on the antigen binding member, or on both. (Unlessotherwise indicated, when members or elements of an RCAR are describedherein, the order can be as provided, but other orders are included aswell. In other words, in an embodiment, the order is as set out in thetext, but in other embodiments, the order can be different. E.g., theorder of elements on one side of a transmembrane region can be differentfrom the example, e.g., the placement of a switch domain relative to aintracellular signaling domain can be different, e.g., reversed).

In an embodiment, the first and second switch domains can form anintracellular or an extracellular dimerization switch. In an embodiment,the dimerization switch can be a homodimerization switch, e.g., wherethe first and second switch domain are the same, or a heterodimerizationswitch, e.g., where the first and second switch domain are differentfrom one another.

In embodiments, an RCAR can comprise a “multi switch.” A multi switchcan comprise heterodimerization switch domains or homodimerizationswitch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member,e.g., an antigen binding member, and a second member, e.g., anintracellular signaling member. In an embodiment, the first member cancomprise a plurality of first switch domains, e.g., FKBP-based switchdomains, and the second member can comprise a plurality of second switchdomains, e.g., FRB-based switch domains. In an embodiment, the firstmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain, and the secondmember can comprise a first and a second switch domain, e.g., aFKBP-based switch domain and a FRB-based switch domain.

In an embodiment, the intracellular signaling member comprises one ormore intracellular signaling domains, e.g., a primary intracellularsignaling domain and one or more costimulatory signaling domains.

In an embodiment, the antigen binding member may comprise one or moreintracellular signaling domains, e.g., one or more costimulatorysignaling domains. In an embodiment, the antigen binding membercomprises a plurality, e.g., 2 or 3 costimulatory signaling domainsdescribed herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40,and in embodiments, no primary intracellular signaling domain. In anembodiment, the antigen binding member comprises the followingcostimulatory signaling domains, from the extracellular to intracellulardirection: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB;OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, theintracellular binding member comprises a CD3zeta domain. In one suchembodiment the RCAR comprises (1) an antigen binding member comprising,an antigen binding domain, a transmembrane domain, and two costimulatorydomains and a first switch domain; and (2) an intracellular signalingdomain comprising a transmembrane domain or membrane tethering domainand at least one primary intracellular signaling domain, and a secondswitch domain.

An embodiment provides RCARs wherein the antigen binding member is nottethered to the surface of the CAR cell. This allows a cell having anintracellular signaling member to be conveniently paired with one ormore antigen binding domains, without transforming the cell with asequence that encodes the antigen binding member. In such embodiments,the RCAR comprises: 1) an intracellular signaling member comprising: afirst switch domain, a transmembrane domain, an intracellular signalingdomain, e.g., a primary intracellular signaling domain, and a firstswitch domain; and 2) an antigen binding member comprising: an antigenbinding domain, and a second switch domain, wherein the antigen bindingmember does not comprise a transmembrane domain or membrane tetheringdomain, and, optionally, does not comprise an intracellular signalingdomain. In some embodiments, the RCAR may further comprise 3) a secondantigen binding member comprising: a second antigen binding domain,e.g., a second antigen binding domain that binds a different antigenthan is bound by the antigen binding domain; and a second switch domain.

Also provided herein are RCARs wherein the antigen binding membercomprises bispecific activation and targeting capacity. In thisembodiment, the antigen binding member can comprise a plurality, e.g.,2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigenbinding domain binds to a target antigen, e.g. different antigens or thesame antigen, e.g., the same or different epitopes on the same antigen.In an embodiment, the plurality of antigen binding domains are intandem, and optionally, a linker or hinge region is disposed betweeneach of the antigen binding domains. Suitable linkers and hinge regionsare described herein.

An embodiment provides RCARs having a configuration that allowsswitching of proliferation. In this embodiment, the RCAR comprises: 1)an intracellular signaling member comprising: optionally, atransmembrane domain or membrane tethering domain; one or moreco-stimulatory signaling domain, e.g., selected from 41BB, CD28, CD27,ICOS, and OX40, and a switch domain; and 2) an antigen binding membercomprising: an antigen binding domain, a transmembrane domain, and aprimary intracellular signaling domain, e.g., a CD3zeta domain, whereinthe antigen binding member does not comprise a switch domain, or doesnot comprise a switch domain that dimerizes with a switch domain on theintracellular signaling member. In an embodiment, the antigen bindingmember does not comprise a co-stimulatory signaling domain. In anembodiment, the intracellular signaling member comprises a switch domainfrom a homodimerization switch. In an embodiment, the intracellularsignaling member comprises a first switch domain of a heterodimerizationswitch and the RCAR comprises a second intracellular signaling memberwhich comprises a second switch domain of the heterodimerization switch.In such embodiments, the second intracellular signaling member comprisesthe same intracellular signaling domains as the intracellular signalingmember. In an embodiment, the dimerization switch is intracellular. Inan embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described here, the first and secondswitch domains comprise a FKBP-FRB based switch as described herein.

Also provided herein are cells comprising an RCAR described herein. Anycell that is engineered to express a RCAR can be used as a RCARX cell.In an embodiment the RCARX cell is a T cell, and is referred to as aRCART cell. In an embodiment the RCARX cell is an NK cell, and isreferred to as a RCARN cell.

Also provided herein are nucleic acids and vectors comprising RCARencoding sequences. Sequence encoding various elements of an RCAR can bedisposed on the same nucleic acid molecule, e.g., the same plasmid orvector, e.g., viral vector, e.g., lentiviral vector. In an embodiment,(i) sequence encoding an antigen binding member and (ii) sequenceencoding an intracellular signaling member, can be present on the samenucleic acid, e.g., vector. Production of the corresponding proteins canbe achieved, e.g., by the use of separate promoters, or by the use of abicistronic transcription product (which can result in the production oftwo proteins by cleavage of a single translation product or by thetranslation of two separate protein products). In an embodiment, asequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, isdisposed between (i) and (ii). In an embodiment, a sequence encoding anIRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). Inthese embodiments, (i) and (ii) are transcribed as a single RNA. In anembodiment, a first promoter is operably linked to (i) and a secondpromoter is operably linked to (ii), such that (i) and (ii) aretranscribed as separate mRNAs.

Alternatively, the sequence encoding various elements of an RCAR can bedisposed on the different nucleic acid molecules, e.g., differentplasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g.,the (i) sequence encoding an antigen binding member can be present on afirst nucleic acid, e.g., a first vector, and the (ii) sequence encodingan intracellular signaling member can be present on the second nucleicacid, e.g., the second vector.

Dimerization Switches

Dimerization switches can be non-covalent or covalent. In a non-covalentdimerization switch, the dimerization molecule promotes a non-covalentinteraction between the switch domains. In a covalent dimerizationswitch, the dimerization molecule promotes a covalent interactionbetween the switch domains.

In an embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB-baseddimerization switch. FKBP12 (FKBP, or FK506 binding protein) is anabundant cytoplasmic protein that serves as the initial intracellulartarget for the natural product immunosuppressive drug, rapamycin.Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR).FRB is a 93 amino acid portion of FRAP, that is sufficient for bindingthe FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. &Schreiber, S. L. (1995) Identification of an 11-kDaFKBP12-rapamycin-binding domain within the 289-kDaFKBP12-rapamycin-associated protein and characterization of a criticalserine residue. Proc Natl Acad Sci USA 92: 4947-51.)

In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based switch can use adimerization molecule, e.g., rapamycin or a rapamycin analog.

The amino acid sequence of FKBP is as follows:

(SEQ ID NO: 52) D V P D Y A S L G G P S S P K K K R K V S R G V Q V E TI S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D RN K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S PD Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y

In embodiments, an FKBP switch domain can comprise a fragment of FKBPhaving the ability to bind with FRB, or a fragment or analog thereof, inthe presence of rapamycin or a rapalog, e.g., the underlined portion ofSEQ ID NO: 52, which is:

(SEQ ID NO: 53) V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G ML E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A QM S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D VE L L K L E T S

The amino acid sequence of FRB is as follows:

(SEQ ID NO: 54) ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSFNQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK

“FKBP/FRAP, e.g., an FKBP/FRB, based switch” as that term is usedherein, refers to a dimerization switch comprising: a first switchdomain, which comprises an FKBP fragment or analog thereof having theability to bind with FRB, or a fragment or analog thereof, in thepresence of rapamycin or a rapalog, e.g., RAD001, and has at least 70,75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by nomore than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from,the FKBP sequence of SEQ ID NO: 52 or 53; and a second switch domain,which comprises an FRB fragment or analog thereof having the ability tobind with FRB, or a fragment or analog thereof, in the presence ofrapamycin or a rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97,98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10,5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ IDNO: 54. In an embodiment, a RCAR described herein comprises one switchdomain comprises amino acid residues disclosed in SEQ ID NO: 52 (or SEQID NO: 53), and one switch domain comprises amino acid residuesdisclosed in SEQ ID NO: 54.

In embodiments, the FKBP/FRB dimerization switch comprises a modifiedFRB switch domain that exhibits altered, e.g., enhanced, complexformation between an FRB-based switch domain, e.g., the modified FRBswitch domain, a FKBP-based switch domain, and the dimerizationmolecule, e.g., rapamycin or a rapalogue, e.g., RAD001. In anembodiment, the modified FRB switch domain comprises one or moremutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected frommutations at amino acid position(s) L2031, E2032, S2035, R2036, F2039,G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type aminoacid is mutated to any other naturally-occurring amino acid. In anembodiment, a mutant FRB comprises a mutation at E2032, where E2032 ismutated to phenylalanine (E2032F), methionine (E2032M), arginine(E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g.,SEQ ID NO: 55, or leucine (E2032L), e.g., SEQ ID NO: 56. In anembodiment, a mutant FRB comprises a mutation at T2098, where T2098 ismutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO:57. In an embodiment, a mutant FRB comprises a mutation at E2032 and atT2098, where E2032 is mutated to any amino acid, and where T2098 ismutated to any amino acid, e.g., SEQ ID NO: 58. In an embodiment, amutant FRB comprises an E20321 and a T2098L mutation, e.g., SEQ ID NO:59. In an embodiment, a mutant FRB comprises an E2032L and a T2098Lmutation, e.g., SEQ ID NO: 60.

TABLE 1D Exemplary mutant FRB having increased affinity for adimerization molecule. SEQ ID FRB mutant Amino Acid Sequence NO: E2032Imutant ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 55QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS E2032L mutantILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 56QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS T2098L mutantILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 57QAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032, T2098 ILWHEMWHEGL XEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 58 mutantQAYGRDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS E2032I, T2098LILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 59 mutantQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032L, T2098LILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN 60 mutantQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS

Other suitable dimerization switches include a GyrB-GyrB baseddimerization switch, a Gibberellin-based dimerization switch, atag/binder dimerization switch, and a halo-tag/snap-tag dimerizationswitch. Following the guidance provided herein, such switches andrelevant dimerization molecules will be apparent to one of ordinaryskill.

Dimerization Molecule

Association between the switch domains is promoted by the dimerizationmolecule. In the presence of dimerization molecule interaction orassociation between switch domains allows for signal transductionbetween a polypeptide associated with, e.g., fused to, a first switchdomain, and a polypeptide associated with, e.g., fused to, a secondswitch domain. In the presence of non-limiting levels of dimerizationmolecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in asystem described herein.

Rapamycin and rapamycin analogs (sometimes referred to as rapalogues),e.g., RAD001, can be used as dimerization molecules in a FKBP/FRB-baseddimerization switch described herein. In an embodiment the dimerizationmolecule can be selected from rapamycin (sirolimus), RAD001(everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus),biolimus and AP21967. Additional rapamycin analogs suitable for use withFKBP/FRB-based dimerization switches are further described in thesection entitled “Combination Therapies”, or in the subsection entitled“Exemplary mTOR inhibitors”.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657. Briefly, a split CAR system comprises a cellexpressing a first CAR having a first antigen binding domain and acostimulatory domain (e.g., 41BB), and the cell also expresses a secondCAR having a second antigen binding domain and an intracellularsignaling domain (e.g., CD3 zeta). When the cell encounters the firstantigen, the costimulatory domain is activated, and the cellproliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. The present invention also includes a CAR encoding RNA constructthat can be directly transfected into a cell. A method for generatingmRNA for use in transfection can involve in vitro transcription (IVT) ofa template with specially designed primers, followed by polyA addition,to produce a construct containing 3′ and 5′ untranslated sequence(“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), thenucleic acid to be expressed, and a polyA tail, typically 50-2000 basesin length (SEQ ID NO:32). RNA so produced can efficiently transfectdifferent kinds of cells. In one aspect, the template includes sequencesfor the CAR.

In one aspect, a CAR of the present invention is encoded by a messengerRNA (mRNA). In one aspect, the mRNA encoding a CAR described herein isintroduced into an immune effector cell, e.g., a T cell or a NK cell,for production of a CAR-expressing cell, e.g., a CART cell or a CAR NKcell.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR described herein. For example, the template forthe RNA CAR comprises an extracellular region comprising a single chainvariable domain of an antibody to a tumor associated antigen describedherein; a hinge region (e.g., a hinge region described herein), atransmembrane domain (e.g., a transmembrane domain described herein suchas a transmembrane domain of CD8a); and a cytoplasmic region thatincludes an intracellular signaling domain, e.g., an intracellularsignaling domain described herein, e.g., comprising the signaling domainof CD3-zeta and the signaling domain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 35) (size can be 50-5000 T (SEQ ID NO: 36)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQID NO: 37).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 38) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3′ end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Non-Viral Delivery Methods

In some aspects, non-viral methods can be used to deliver a nucleic acidencoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of atransposon (also called a transposable element). In some embodiments, atransposon is a piece of DNA that can insert itself at a location in agenome, for example, a piece of DNA that is capable of self-replicatingand inserting its copy into a genome, or a piece of DNA that can bespliced out of a longer nucleic acid and inserted into another place ina genome. For example, a transposon comprises a DNA sequence made up ofinverted repeats flanking genes for transposition.

Exemplary methods of nucleic acid delivery using a transposon include aSleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposonsystem. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15 (2008):2961-2971; Huang etal. Mol. Ther. 16 (2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21 (2013):166; Williams.Molecular Therapy 16.9 (2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3 (2005):473-83, all of whichare incorporated herein by reference.

The SBTS includes two components: 1) a transposon containing a transgeneand 2) a source of transposase enzyme. The transposase can transpose thetransposon from a carrier plasmid (or other donor DNA) to a target DNA,such as a host cell chromosome/genome. For example, the transposasebinds to the carrier plasmid/donor DNA, cuts the transposon (includingtransgene(s)) out of the plasmid, and inserts it into the genome of thehost cell. See, e.g., Aronovich et al. supra.

Exemplary transposons include a pT2-based transposon. See, e.g.,Grabundzija et al. Nucleic Acids Res. 41.3 (2013):1829-47; and Singh etal. Cancer Res. 68.8 (2008): 2961-2971, all of which are incorporatedherein by reference. Exemplary transposases include a Tc1/mariner-typetransposase, e.g., the SB10 transposase or the SB11 transposase (ahyperactive transposase which can be expressed, e.g., from acytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.;and Grabundzija et al., all of which are incorporated herein byreference.

Use of the SBTS permits efficient integration and expression of atransgene, e.g., a nucleic acid encoding a CAR described herein.Provided herein are methods of generating a cell, e.g., T cell or NKcell, that stably expresses a CAR described herein, e.g., using atransposon system such as SBTS.

In accordance with methods described herein, in some embodiments, one ormore nucleic acids, e.g., plasmids, containing the SBTS components aredelivered to a cell (e.g., T or NK cell). For example, the nucleicacid(s) are delivered by standard methods of nucleic acid (e.g., plasmidDNA) delivery, e.g., methods described herein, e.g., electroporation,transfection, or lipofection. In some embodiments, the nucleic acidcontains a transposon comprising a transgene, e.g., a nucleic acidencoding a CAR described herein. In some embodiments, the nucleic acidcontains a transposon comprising a transgene (e.g., a nucleic acidencoding a CAR described herein) as well as a nucleic acid sequenceencoding a transposase enzyme. In other embodiments, a system with twonucleic acids is provided, e.g., a dual-plasmid system, e.g., where afirst plasmid contains a transposon comprising a transgene, and a secondplasmid contains a nucleic acid sequence encoding a transposase enzyme.For example, the first and the second nucleic acids are co-deliveredinto a host cell.

In some embodiments, cells, e.g., T or NK cells, are generated thatexpress a CAR described herein by using a combination of gene insertionusing the SBTS and genetic editing using a nuclease (e.g., Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, or engineered meganucleasere-engineered homing endonucleases).

In some embodiments, use of a non-viral method of delivery permitsreprogramming of cells, e.g., T or NK cells, and direct infusion of thecells into a subject. Advantages of non-viral vectors include but arenot limited to the ease and relatively low cost of producing sufficientamounts required to meet a patient population, stability during storage,and lack of immunogenicity.

Nucleic Acid Constructs Encoding a CAR

The present invention also provides nucleic acid molecules encoding oneor more CAR constructs described herein. In one aspect, the nucleic acidmolecule is provided as a messenger RNA transcript. In one aspect, thenucleic acid molecule is provided as a DNA construct.

Accordingly, in one aspect, the invention pertains to a nucleic acidmolecule encoding a chimeric antigen receptor (CAR), wherein the CARcomprises an antigen binding domain that binds to a tumor antigendescribed herein, a transmembrane domain (e.g., a transmembrane domaindescribed herein), and an intracellular signaling domain (e.g., anintracellular signaling domain described herein) comprising astimulatory domain, e.g., a costimulatory signaling domain (e.g., acostimulatory signaling domain described herein) and/or a primarysignaling domain (e.g., a primary signaling domain described herein,e.g., a zeta chain described herein). In one embodiment, thetransmembrane domain is transmembrane domain of a protein selected fromthe group consisting of the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, atransmembrane domain may include at least the transmembrane region(s)of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278),4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1),NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1,VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d,ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1,CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp.

In one embodiment, the transmembrane domain comprises a sequence of SEQID NO: 12, or a sequence with 95-99% identity thereof. In oneembodiment, the antigen binding domain is connected to the transmembranedomain by a hinge region, e.g., a hinge described herein. In oneembodiment, the hinge region comprises SEQ ID NO:4 or SEQ ID NO:6 or SEQID NO:8 or SEQ ID NO:10, or a sequence with 95-99% identity thereof. Inone embodiment, the isolated nucleic acid molecule further comprises asequence encoding a costimulatory domain. In one embodiment, thecostimulatory domain is a functional signaling domain of a proteinselected from the group consisting of OX40, CD27, CD28, CD5, ICAM-1,LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples ofsuch costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, and PAG/Cbp. In one embodiment, thecostimulatory domain comprises a sequence of SEQ ID NO:16, or a sequencewith 95-99% identity thereof. In one embodiment, the intracellularsignaling domain comprises a functional signaling domain of 4-1BB and afunctional signaling domain of CD3 zeta. In one embodiment, theintracellular signaling domain comprises the sequence of SEQ ID NO: 14or SEQ ID NO:16, or a sequence with 95-99% identity thereof, and thesequence of SEQ ID NO: 18 or SEQ ID NO:20, or a sequence with 95-99%identity thereof, wherein the sequences comprising the intracellularsignaling domain are expressed in the same frame and as a singlepolypeptide chain.

In another aspect, the invention pertains to an isolated nucleic acidmolecule encoding a CAR construct comprising a leader sequence of SEQ IDNO: 2, a scFv domain as described herein, a hinge region of SEQ ID NO:4or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 (or a sequence with 95-99%identity thereof), a transmembrane domain having a sequence of SEQ IDNO: 12 (or a sequence with 95-99% identity thereof), a 4-1BBcostimulatory domain having a sequence of SEQ ID NO:14 or a CD27costimulatory domain having a sequence of SEQ ID NO:16 (or a sequencewith 95-99% identity thereof), and a CD3 zeta stimulatory domain havinga sequence of SEQ ID NO:18 or SEQ ID NO:20 (or a sequence with 95-99%identity thereof).

In another aspect, the invention pertains to a nucleic acid moleculeencoding a chimeric antigen receptor (CAR) molecule that comprises anantigen binding domain, a transmembrane domain, and an intracellularsignaling domain comprising a stimulatory domain, and wherein saidantigen binding domain binds to a tumor antigen selected from a groupconsisting of: CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1),CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72,CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra,PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptoralpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PRSS21, PAP, ELF2M,Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase,EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folatereceptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97,CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1,ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a,MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17,XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, andIGLL1.

In one embodiment, the encoded CAR molecule further comprises a sequenceencoding a costimulatory domain. In one embodiment, the costimulatorydomain is a functional signaling domain of a protein selected from thegroup consisting of OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18)and 4-1BB (CD137). In one embodiment, the costimulatory domain comprisesa sequence of SEQ ID NO:14. In one embodiment, the transmembrane domainis a transmembrane domain of a protein selected from the groupconsisting of the alpha, beta or zeta chain of the T-cell receptor,CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37,CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, thetransmembrane domain comprises a sequence of SEQ ID NO:12. In oneembodiment, the intracellular signaling domain comprises a functionalsignaling domain of 4-1BB and a functional signaling domain of zeta. Inone embodiment, the intracellular signaling domain comprises thesequence of SEQ ID NO: 14 and the sequence of SEQ ID NO: 18, wherein thesequences comprising the intracellular signaling domain are expressed inthe same frame and as a single polypeptide chain. In one embodiment, theanti-a cancer associated antigen as described herein binding domain isconnected to the transmembrane domain by a hinge region. In oneembodiment, the hinge region comprises SEQ ID NO:4. In one embodiment,the hinge region comprises SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. A retroviral vector may also be, e.g., a gammaretroviralvector. A gammaretroviral vector may include, e.g., a promoter, apackaging signal (ψ), a primer binding site (PBS), one or more (e.g.,two) long terminal repeats (LTR), and a transgene of interest, e.g., agene encoding a CAR. A gammaretroviral vector may lack viral structuralgens such as gag, pol, and env. Exemplary gammaretroviral vectorsinclude Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV),and Myeloproliferative Sarcoma Virus (MPSV), and vectors derivedtherefrom. Other gammaretroviral vectors are described, e.g., in TobiasMaetzig et al., “Gammaretroviral Vectors: Biology, Technology andApplication” Viruses. 2011 June; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding CARs can beaccomplished using of transposons such as sleeping beauty, crisper,CAS9, and zinc finger nucleases. See below June et al. 2009 NatureReviews Immunology 9.10: 704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription. Exemplary promoters include theCMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK)promoters.

An example of a promoter that is capable of expressing a CAR encodingnucleic acid molecule in a mammalian T cell is the EF1a promoter. Thenative EF1a promoter drives expression of the alpha subunit of theelongation factor-1 complex, which is responsible for the enzymaticdelivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has beenextensively used in mammalian expression plasmids and has been shown tobe effective in driving CAR expression from nucleic acid moleculescloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther.17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises thesequence provided as SEQ ID NO:1.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto.

However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the elongationfactor-1α promoter, the hemoglobin promoter, and the creatine kinasepromoter. Further, the invention should not be limited to the use ofconstitutive promoters. Inducible promoters are also contemplated aspart of the invention. The use of an inducible promoter provides amolecular switch capable of turning on expression of the polynucleotidesequence which it is operatively linked when such expression is desired,or turning off the expression when expression is not desired. Examplesof inducible promoters include, but are not limited to a metallothioninepromoter, a glucocorticoid promoter, a progesterone promoter, and atetracycline promoter.

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection (e.g.,ampicillin resistance gene and/or zeocin marker).

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a CARencoding nucleic acid molecule. In one aspect, a CAR vector can bedirectly transduced into a cell, e.g., a T cell or a NK cell. In oneaspect, the vector is a cloning or expression vector, e.g., a vectorincluding, but not limited to, one or more plasmids (e.g., expressionplasmids, cloning vectors, minicircles, minivectors, double minutechromosomes), retroviral and lentiviral vector constructs. In oneaspect, the vector is capable of expressing the CAR construct inmammalian immune effector cells (e.g., T cells, NK cells). In oneaspect, the mammalian T cell is a human T cell. In one aspect, themammalian NK cell is a human NK cell.

Sources of Cells

Prior to expansion and genetic modification or other modification, asource of cells, e.g., T cells or natural killer (NK) cells, can beobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, monkeys, chimpanzees,dogs, cats, mice, rats, and transgenic species thereof. T cells can beobtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors.

In certain aspects of the present disclosure, immune effector cells,e.g., T cells, can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred aspect, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and, optionally,to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one embodiment, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations.

Initial activation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. Preferably, thepopulation of T regulatory depleted cells contains less than 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody,or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1×10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory depleted cells has2×10⁹ T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹,5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of subject relapse. For example, methods of depletingT_(REG) cells are known in the art. Methods of decreasing T_(REG) cellsinclude, but are not limited to, cyclophosphamide, anti-GITR antibody(an anti-GITR antibody described herein), CD25-depletion, andcombinations thereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a subject is pre-treated with one or more therapiesthat reduce T_(REG) cells prior to collection of cells forCAR-expressing cell product manufacturing, thereby reducing the risk ofsubject relapse to CAR-expressing cell treatment. In an embodiment,methods of decreasing T_(REG) cells include, but are not limited to,administration to the subject of one or more of cyclophosphamide,anti-GITR antibody, CD25-depletion, or a combination thereof.Administration of one or more of cyclophosphamide, anti-GITR antibody,CD25-depletion, or a combination thereof, can occur before, during orafter an infusion of the CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment. In an embodiment, a subject is pre-treated with an anti-GITRantibody prior to collection of cells for CAR-expressing cell productmanufacturing, thereby reducing the risk of subject relapse toCAR-expressing cell treatment.

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of T regulatorydepleted, e.g., CD25+ depleted cells, and check point inhibitor depletedcells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLAand LAIR1. In one embodiment, check point inhibitor expressing cells areremoved simultaneously with the T regulatory, e.g., CD25+ cells. Forexample, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to thesame bead which can be used to remove the cells, or an anti-CD25antibody, or fragment thereof, and the anti-check point inhibitorantibody, or fragment there, can be attached to separate beads, amixture of which can be used to remove the cells. In other embodiments,the removal of T regulatory cells, e.g., CD25+ cells, and the removal ofthe check point inhibitor expressing cells is sequential, and can occur,e.g., in either order.

Methods described herein can include a positive selection step. Forexample, T cells can isolated by incubation with anti-CD3/anti-CD28(e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, fora time period sufficient for positive selection of the desired T cells.In one embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment,the time period is 10 to 24 hours, e.g., 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In yet one aspect, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtheraspects, concentrations of 125 or 150 million cells/ml can be used.

Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in immune effector cell therapy for any number of diseasesor conditions that would benefit from immune effector cell therapy, suchas those described herein. In one aspect a blood sample or an apheresisis taken from a generally healthy subject. In certain aspects, a bloodsample or an apheresis is taken from a generally healthy subject who isat risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In certain aspects, the T cells may be expanded, frozen, and usedat a later time. In certain aspects, samples are collected from apatient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further aspect, the cells areisolated from a blood sample or an apheresis from a subject prior to anynumber of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diacylglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression.

Alternatively, Ikaros-deficient cells can be generated by treatment withIkaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Allogeneic CAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of a functional T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCRor engineered such that it produces very little functional TCR on itssurface. Alternatively, the T cell can express a substantially impairedTCR, e.g., by expression of mutated or truncated forms of one or more ofthe subunits of the TCR. The term “substantially impaired TCR” meansthat this TCR will not elicit an adverse immune reaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class I and/or HLA class II, is downregulated.

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpress or expresses at low levels an inhibitory molecule, e.g. by anymethod described herein. For example, the cell can be a cell that doesnot express or expresses at low levels an inhibitory molecule, e.g.,that can decrease the ability of a CAR-expressing cell to mount animmune effector response. Examples of inhibitory molecules include PD1,PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5),LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition ofan inhibitory molecule, e.g., by inhibition at the DNA, RNA or proteinlevel, can optimize a CAR-expressing cell performance. In embodiments,an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA in a T cell.

Expression of siRNA and shRNAs in T cells can be achieved using anyconventional expression system, e.g., such as a lentiviral expressionsystem.

Exemplary shRNAs that downregulate expression of components of the TCRare described, e.g., in US Publication No.: 2012/0321667. ExemplarysiRNA and shRNA that downregulate expression of HLA class I and/or HLAclass II genes are described, e.g., in U.S. publication No.: US2007/0036773.

CRISPR to Inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene.

Naturally-occurring CRISPR/Cas systems are found in approximately 40% ofsequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al.(2007) BMC Bioinformatics 8: 172. This system is a type of prokaryoticimmune system that confers resistance to foreign genetic elements suchas plasmids and phages and provides a form of acquired immunity.Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008)Science 322: 1843-1845.

The CRISPR/Cas system has been modified for use in gene editing(silencing, enhancing or changing specific genes) in eukaryotes such asmice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This isaccomplished by introducing into the eukaryotic cell a plasmidcontaining a specifically designed CRISPR and one or more appropriateCas.

The CRISPR sequence, sometimes called a CRISPR locus, comprisesalternating repeats and spacers. In a naturally-occurring CRISPR, thespacers usually comprise sequences foreign to the bacterium such as aplasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, thespacers are derived from the TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed byCas proteins into small RNAs. These comprise a spacer flanked by arepeat sequence. The RNAs guide other Cas proteins to silence exogenousgenetic elements at the RNA or DNA level. Horvath et al. (2010) Science327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacersthus serve as templates for RNA molecules, analogously to siRNAs.Pennisi (2013) Science 341: 833-836.

As these naturally occur in many different types of bacteria, the exactarrangements of the CRISPR and structure, function and number of Casgenes and their product differ somewhat from species to species. Haft etal. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol.8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al.(2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. Forexample, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. Brouns et al. (2008) Science321: 960-964. In other prokaryotes, Cas6 processes the CRISPRtranscript. The CRISPR-based phage inactivation in E. coli requiresCascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module)proteins in Pyrococcus furiosus and other prokaryotes form a functionalcomplex with small CRISPR RNAs that recognizes and cleaves complementarytarget RNAs. A simpler CRISPR system relies on the protein Cas9, whichis a nuclease with two active cutting sites, one for each strand of thedouble helix. Combining Cas9 and modified CRISPR locus RNA can be usedin a system for gene editing. Pennisi (2013) Science 341: 833-836.

The CRISPR/Cas system can thus be used to edit a TCR and/or HLA gene(adding or deleting a basepair), or introducing a premature stop whichthus decreases expression of a TCR and/or HLA. The CRISPR/Cas system canalternatively be used like RNA interference, turning off TCR and/or HLAgene in a reversible fashion. In a mammalian cell, for example, the RNAcan guide the Cas protein to a TCR and/or HLA promoter, stericallyblocking RNA polymerases.

Artificial CRISPR/Cas systems can be generated which inhibit TCR and/orHLA, using technology known in the art, e.g., that described in U.S.Publication No. 20140068797, and Cong (2013) Science 339: 819-823. Otherartificial CRISPR/Cas systems that are known in the art may also begenerated which inhibit TCR and/or HLA, e.g., that described in Tsai(2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445;8,865,406; 8,795,965; 8,771,945; and 8,697,359.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene.

TALENs are produced artificially by fusing a TAL effector DNA bindingdomain to a DNA cleavage domain. Transcription activator-like effects(TALEs) can be engineered to bind any desired DNA sequence, including aportion of the HLA or TCR gene. By combining an engineered TALE with aDNA cleavage domain, a restriction enzyme can be produced which isspecific to any desired DNA sequence, including a HLA or TCR sequence.These can then be introduced into a cell, wherein they can be used forgenome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al.(2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

TALEs are proteins secreted by Xanthomonas bacteria. The DNA bindingdomain contains a repeated, highly conserved 33-34 amino acid sequence,with the exception of the 12th and 13th amino acids. These two positionsare highly variable, showing a strong correlation with specificnucleotide recognition. They can thus be engineered to bind to a desiredDNA sequence.

To produce a TALEN, a TALE protein is fused to a nuclease (N), which isa wild-type or mutated Fold endonuclease. Several mutations to FokI havebeen made for its use in TALENs; these, for example, improve cleavagespecificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82;Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011)Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyonet al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) NatureBiotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.

The FokI domain functions as a dimer, requiring two constructs withunique DNA binding domains for sites in the target genome with properorientation and spacing. Both the number of amino acid residues betweenthe TALE DNA binding domain and the FokI cleavage domain and the numberof bases between the two individual TALEN binding sites appear to beimportant parameters for achieving high levels of activity. Miller etal. (2011) Nature Biotech. 29: 143-8.

A HLA or TCR TALEN can be used inside a cell to produce adouble-stranded break (DSB). A mutation can be introduced at the breaksite if the repair mechanisms improperly repair the break vianon-homologous end joining. For example, improper repair may introduce aframe shift mutation. Alternatively, foreign DNA can be introduced intothe cell along with the TALEN; depending on the sequences of the foreignDNA and chromosomal sequence, this process can be used to correct adefect in the HLA or TCR gene or introduce such a defect into a wt HLAor TCR gene, thus decreasing expression of HLA or TCR.

TALENs specific to sequences in HLA or TCR can be constructed using anymethod known in the art, including various schemes using modularcomponents. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler etal. (2011) PLoS ONE 6: e19509.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene.

Like a TALEN, a ZFN comprises a Fold nuclease domain (or derivativethereof) fused to a DNA-binding domain. In the case of a ZFN, theDNA-binding domain comprises one or more zinc fingers. Carroll et al.(2011) Genetics Society of America 188: 773-782; and Kim et al. (1996)Proc. Natl. Acad. Sci. USA 93: 1156-1160.

A zinc finger is a small protein structural motif stabilized by one ormore zinc ions. A zinc finger can comprise, for example, Cys2His2, andcan recognize an approximately 3-bp sequence. Various zinc fingers ofknown specificity can be combined to produce multi-finger polypeptideswhich recognize about 6, 9, 12, 15 or 18-bp sequences. Various selectionand modular assembly techniques are available to generate zinc fingers(and combinations thereof) recognizing specific sequences, includingphage display, yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNsare required to target non-palindromic DNA sites. The two individualZFNs must bind opposite strands of the DNA with their nucleases properlyspaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

Also like a TALEN, a ZFN can create a double-stranded break in the DNA,which can create a frame-shift mutation if improperly repaired, leadingto a decrease in the expression and amount of HLA and/or TCR in a cell.ZFNs can also be used with homologous recombination to mutate in the HLAor TCR gene.

ZFNs specific to sequences in HLA AND/OR TCR can be constructed usingany method known in the art. See, e.g., Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008)Mol. Ther. 16: 1200-7; Guo et al. (2010) J. Mol. Biol. 400: 96; U.S.Patent Publication 2011/0158957; and U.S. Patent Publication2012/0060230.

Telomerase Expression

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

In one aspect, the disclosure features a method of making a populationof immune effector cells (e.g., T cells, NK cells). In an embodiment,the method comprises: providing a population of immune effector cells(e.g., T cells or NK cells), contacting the population of immuneeffector cells with a nucleic acid encoding a CAR; and contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT, under conditions that allow for CAR andtelomerase expression.

In an embodiment, the nucleic acid encoding the telomerase subunit isDNA. In an embodiment, the nucleic acid encoding the telomerase subunitcomprises a promoter capable of driving expression of the telomerasesubunit.

In an embodiment, hTERT has the amino acid sequence of GenBank ProteinID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human TelomeraseCatalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795)as follows:

(SEQ ID NO: 61) MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%,96^(∧), 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 61. Inan embodiment, the hTERT has a sequence of SEQ ID NO: 61. In anembodiment, the hTERT comprises a deletion (e.g., of no more than 5, 10,15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both.In an embodiment, the hTERT comprises a transgenic amino acid sequence(e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at theN-terminus, the C-terminus, or both. In an embodiment, the hTERT isencoded by the nucleic acid sequence of GenBank Accession No. AF018167(Meyerson et al., “hEST2, the Putative Human Telomerase CatalyticSubunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization”Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795):

(SEQ ID NO: 62) 1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacccccgcgatgc 61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactaccgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctggtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtgccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaaggagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggccttcggcttcg 361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagcgtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtgggggctgctgttgc 481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctctttgtgctgg 541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctcggcgctgcca 601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctgggatgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagccccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggcccaggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccacccgggcagga 841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagacccgccgaagaag 901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatccgtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggacacgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaaggagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcggaggctcgtgg 1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcaggttgccccgcc 1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttgggaaccacgcgc 1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcggtcaccccag 1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggcccccgaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccctggcaggtgt 1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctggggctccaggc 1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctggggaagcatgcca 1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgcttggctgcgca 1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgaggagatcctgg 1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcaggtctttctttt 1741 atgtcacgga gaccacgat caaaagaaca ggctcttttt ctaccggaagagtgtctgga 1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctgcgggagctgt 1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacgtccagactcc 1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactacgtcgtgggag 1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtgaaggcactgt 2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcctctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgggcccaggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgacaccatccccc 2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacgtactgcgtgc 2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggccttcaagagcc 2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggctcacctgcagg 2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctgaatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtgcgcatcaggg 2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctccacgctgctct 2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcggcgggacgggc 2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacccacgcgaaaa 2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtgaacttgcgga 2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggcttttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccggaccctggagg 2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctcaccttcaacc 2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttgcggctgaagt 3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgcaccaacatct 3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcagctcccatttc 3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgacacggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggccaagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattcctgctcaagc 3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggacagcccagacgc 3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgcagccaacccgg 3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccacagccaggccg 3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagggaggggcggc 3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttggccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtccagccaagggc 3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcggctccacccc 3721 agggccagct tacctcacc aggagcccgg cttccactcc ccacataggaatagtccatc 3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccacccccaccatc 3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtgaccaaaggtgt 3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggtcaaattgggg 3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttgaaaaaaaaaaa 4021 aaaaaaa

In an embodiment, the hTERT is encoded by a nucleic acid having asequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 62. In an embodiment, the hTERT is encoded bya nucleic acid of SEQ ID NO: 62.

Activation and Expansion of Immune Effector Cells (e.g., T Cells)

Immune effector cells such as T cells may be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005.

As demonstrated by the data disclosed herein, expanding the T cells bythe methods disclosed herein can multiply the cells by about 10 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold,6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold,1,000,000 fold, 10,000,000 fold, or greater, and any and all whole orpartial integers therebetween. In one embodiment, the T cells expand inthe range of about 20 fold to about 50 fold.

Generally, a population of immune effector cells e.g., T regulatory celldepleted cells, may be expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a costimulatory molecule on thesurface of the T cells. In particular, T cell populations may bestimulated as described herein, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibodycan be used. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect, more anti-CD28 antibody is bound tothe particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 isless than one. In certain aspects, the ratio of anti CD28 antibody toanti CD3 antibody bound to the beads is greater than 2:1. In oneparticular aspect, a 1:100 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads isused. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound tobeads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio ofantibody bound to the beads is used. In yet one aspect, a 3:1 CD3:CD28ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain preferredvalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one preferred ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a preferred particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined withagent-coated beads, the beads and the cells are subsequently separated,and then the cells are cultured. In an alternative aspect, prior toculture, the agent-coated beads and cells are not separated but arecultured together. In a further aspect, the beads and cells are firstconcentrated by application of a force, such as a magnetic force,resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded in culture for aperiod of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18,21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In one embodiment, the cells are expanded for a periodof 4 to 9 days. In one embodiment, the cells are expanded for a periodof 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cells,e.g., a CD19 CAR cell described herein, are expanded in culture for 5days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.Potency can be defined, e.g., by various T cell functions, e.g.proliferation, target cell killing, cytokine production, activation,migration, or combinations thereof. In one embodiment, the cells, e.g.,a CD19 CAR cell described herein, expanded for 5 days show at least aone, two, three or four fold increase in cells doublings upon antigenstimulation as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,the cells expressing a CD19 CAR described herein, are expanded inculture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., a CD19 CARcell described herein, expanded for 5 days show at least a one, two,three, four, five, ten fold or more increase in pg/ml of proinflammatorycytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared tothe same cells expanded in culture for 9 days under the same cultureconditions.

Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any otheradditives for the growth of cells known to the skilled artisan. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of T cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence of IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetIL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a CAR described herein is constructed, various assays can be usedto evaluate the activity of the molecule, such as but not limited to,the ability to expand T cells following antigen stimulation, sustain Tcell expansion in the absence of re-stimulation, and anti-canceractivities in appropriate in vitro and animal models. Assays to evaluatethe effects of a cars of the present invention are described in furtherdetail below

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers. See, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1mixture of CD4⁺ and CD8⁺ T cells) expressing the CARs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. CARs containing the full length TCR-ζ cytoplasmicdomain and the endogenous TCR-ζ chain are detected by western blottingusing an antibody to the TCR-ζ chain. The same T cell subsets are usedfor SDS-PAGE analysis under non-reducing conditions to permit evaluationof covalent dimer formation.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either a cancer associated antigen asdescribed herein⁺ K562 cells (K562 expressing a cancer associatedantigen as described herein), wild-type K562 cells (K562 wild type) orK562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 andanti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous IL-2 isadded to the cultures every other day at 100 IU/ml. GFP⁺ T cells areenumerated by flow cytometry using bead-based counting. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CART activity. For example,xenograft model using human a cancer associated antigen describedherein-specific CAR⁺ T cells to treat a primary human pre-B ALL inimmunodeficient mice can be used. See, e.g., Milone et al., MolecularTherapy 17(8): 1453-1464 (2009). Very briefly, after establishment ofALL, mice are randomized as to treatment groups. Different numbers of acancer associated antigen-specific CAR engineered T cells are coinjectedat a 1:1 ratio into NOD-SCID-γ^(−/−) mice bearing B-ALL. The number ofcopies of a cancer associated antigen-specific CAR vector in spleen DNAfrom mice is evaluated at various times following T cell injection.Animals are assessed for leukemia at weekly intervals. Peripheral blooda cancer associate antigen as described herein⁺ B-ALL blast cell countsare measured in mice that are injected with a cancer associated antigendescribed herein-ζ CAR⁺ T cells or mock-transduced T cells. Survivalcurves for the groups are compared using the log-rank test. In addition,absolute peripheral blood CD4⁺ and CD8⁺ T cell counts 4 weeks followingT cell injection in NOD-SCID-γ^(−/−) mice can also be analyzed. Mice areinjected with leukemic cells and 3 weeks later are injected with T cellsengineered to express CAR by a bicistronic lentiviral vector thatencodes the CAR linked to eGFP. T cells are normalized to 45-50% inputGFP⁺ T cells by mixing with mock-transduced cells prior to injection,and confirmed by flow cytometry. Animals are assessed for leukemia at1-week intervals. Survival curves for the CAR⁺ T cell groups arecompared using the log-rank test.

Dose dependent CAR treatment response can be evaluated. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example,peripheral blood is obtained 35-70 days after establishing leukemia inmice injected on day 21 with CAR T cells, an equivalent number ofmock-transduced T cells, or no T cells. Mice from each group arerandomly bled for determination of peripheral blood a cancer associateantigen as described herein⁺ ALL blast counts and then killed on days 35and 49. The remaining animals are evaluated on days 57 and 70.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation isperformed in microtiter plates by mixing washed T cells with K562 cellsexpressing a cancer associated antigen described herein (K19) or CD32and CD137 (KT32-BBL) for a final T-cell:K562 ratio of 2:1. K562 cellsare irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3)and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultureswith KT32-BBL cells to serve as a positive control for stimulatingT-cell proliferation since these signals support long-term CD8⁺ T cellexpansion ex vivo. T cells are enumerated in cultures using CountBright™fluorescent beads (Invitrogen, Carlsbad, Calif.) and flow cytometry asdescribed by the manufacturer. CAR⁺ T cells are identified by GFPexpression using T cells that are engineered with eGFP-2A linkedCAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP,the CAR+ T cells are detected with biotinylated recombinant a cancerassociate antigen as described herein protein and a secondary avidin-PEconjugate. CD4+ and CD8⁺ expression on T cells are also simultaneouslydetected with specific monoclonal antibodies (BD Biosciences). Cytokinemeasurements are performed on supernatants collected 24 hours followingre-stimulation using the human TH1/TH2 cytokine cytometric bead arraykit (BD Biosciences, San Diego, Calif.) according the manufacturer'sinstructions. Fluorescence is assessed using a FACScalibur flowcytometer, and data is analyzed according to the manufacturer'sinstructions.

Cytotoxicity can be assessed by a standard 51Cr-release assay. See,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly,target cells (K562 lines and primary pro-B-ALL cells) are loaded with51Cr (as NaCrO4, New England Nuclear, Boston, Mass.) at 37° C. for 2hours with frequent agitation, washed twice in complete RPMI and platedinto microtiter plates. Effector T cells are mixed with target cells inthe wells in complete RPMI at varying ratios of effector cell:targetcell (E:T). Additional wells containing media only (spontaneous release,SR) or a 1% solution of triton-X 100 detergent (total release, TR) arealso prepared. After 4 hours of incubation at 37° C., supernatant fromeach well is harvested. Released 51Cr is then measured using a gammaparticle counter (Packard Instrument Co., Waltham, Mass.). Eachcondition is performed in at least triplicate, and the percentage oflysis is calculated using the formula: % Lysis=(ER−SR)/(TR−SR), where ERrepresents the average 51Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models. Such assays havebeen described, for example, in Barrett et al., Human Gene Therapy22:1575-1586 (2011). Briefly, NOD/SCID/γc^(−/−) (NSG) mice are injectedIV with Nalm-6 cells followed 7 days later with T cells 4 hour afterelectroporation with the CAR constructs. The T cells are stablytransfected with a lentiviral construct to express firefly luciferase,and mice are imaged for bioluminescence. Alternatively, therapeuticefficacy and specificity of a single injection of CAR⁺ T cells in Nalm-6xenograft model can be measured as the following: NSG mice are injectedwith Nalm-6 transduced to stably express firefly luciferase, followed bya single tail-vein injection of T cells electroporated with cars of thepresent invention 7 days later. Animals are imaged at various timepoints post injection. For example, photon-density heat maps of fireflyluciferase positive leukemia in representative mice at day 5 (2 daysbefore treatment) and day 8 (24 hr post CAR⁺ PBLs) can be generated.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theCARs described herein.

Therapeutic Application

The modified cells described herein may be included in a composition fortherapy. In one aspect, the composition comprises a population ofmodified T cells comprising a nucleic acid sequence encoding a CAR and anucleic acid sequence encoding a peptide comprising an amphipathic helixdomain and a cluster of basic amino acids, wherein the peptide disruptsPKA and an AKAP association as described herein. In another aspect, thecomposition comprises the modified T cell comprising a nucleic acidsequence encoding a CAR and a nucleic acid sequence encoding a peptidecomprising an amphipathic helix domain and a cluster of basic aminoacids, wherein the peptide disrupts PKA and an AKAP association asdescribed herein. In yet another embodiment, the composition includes amodified T cell comprising a CAR and a peptide described herein, e.g., apeptide that disrupts PKA and an AKAP binding. The composition mayinclude a pharmaceutical composition and further include apharmaceutically acceptable carrier. A therapeutically effective amountof the pharmaceutical composition comprising the modified cells may beadministered.

In one aspect, the invention includes a method comprising administeringa population of modified T cells to a subject in need thereof to preventor treat a tumor, wherein the modified T cells comprise a nucleic acidsequence encoding a CAR and a nucleic acid sequence encoding a peptidedescribed herein, e.g., a peptide comprising an amphipathic helix domainand a cluster of basic amino acids, wherein the peptide disrupts PKA andan AKAP association.

In another aspect, the invention includes a method comprisingadministering a population of modified cells to a subject in needthereof to prevent or treat a tumor that is adverse to the subject,wherein the modified cells comprise a CAR and a peptide describedherein, e.g., a peptide that disrupts PKA and an AKAP binding.

In one aspect, the invention provides methods for treating a diseaseassociated with expression of a cancer associated antigen describedherein.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an XCAR, wherein Xrepresents a tumor antigen as described herein, and wherein the cancercells express said X tumor antigen.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a XCAR describedherein, wherein the cancer cells express X. In one embodiment,

X is expressed on both normal cells and cancers cells, but is expressedat lower levels on normal cells. In one embodiment, the method furthercomprises selecting a CAR that binds X with an affinity that allows theXCAR to bind and kill the cancer cells expressing X but less than 30%,25%, 20%, 15%, 10%, 5% or less of the normal cells expressing X arekilled, e.g., as determined by an assay described herein. In oneembodiment, the selected CAR has an antigen binding domain that has abinding affinity KD of 10⁻⁴ M to 10⁻⁸ M, e.g., 10⁻⁵ M to 10⁻⁷ M, e.g.,10⁻⁶ M or 10⁻⁷ M, for the target antigen. In one embodiment, theselected antigen binding domain has a binding affinity that is at leastfive-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-foldless than a reference antibody, e.g., an antibody described herein.

In one embodiment, the present invention provides methods of treatingcancer by providing to the subject in need thereof immune effector cells(e.g., T cells, NK cells) that are engineered to express CD19 CAR,wherein the cancer cells express CD19. In one embodiment, the cancer tobe treated is ALL (acute lymphoblastic leukemia), CLL (chroniclymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL(Mantle cell lymphoma, or MM (multiple myeloma).

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an EGFRvIIICAR,wherein the cancer cells express EGFRvIII. In one embodiment, the cancerto be treated is glioblastoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a mesothelin CAR,wherein the cancer cells express mesothelin. In one embodiment, thecancer to be treated is mesothelioma, pancreatic cancer, or ovariancancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD123CAR, whereinthe cancer cells express CD123. In one embodiment, the cancer to betreated is AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD22CAR, wherein thecancer cells express CD22. In one embodiment, the cancer to be treatedis B cell malignancies.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CS-1CAR, wherein thecancer cells express CS-1. In one embodiment, the cancer to be treatedis multiple myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CLL-1CAR, whereinthe cancer cells express CLL-1. In one embodiment, the cancer to betreated is AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD33CAR, wherein thecancer cells express CD33. In one embodiment, the cancer to be treatedis AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GD2CAR, wherein thecancer cells express GD2. In one embodiment, the cancer to be treated isneuroblastoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a BCMACAR, wherein thecancer cells express BCMA. In one embodiment, the cancer to be treatedis multiple myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TnCAR, wherein thecancer cells express Tn antigen. In one embodiment, the cancer to betreated is ovarian cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PSMACAR, wherein thecancer cells express PSMA. In one embodiment, the cancer to be treatedis prostate cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a ROR1CAR, wherein thecancer cells express ROR1. In one embodiment, the cancer to be treatedis B cell malignancies.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a FLT3 CAR, whereinthe cancer cells express FLT3. In one embodiment, the cancer to betreated is AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TAG72CAR, whereinthe cancer cells express TAG72. In one embodiment, the cancer to betreated is gastrointestinal cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD38CAR, wherein thecancer cells express CD38. In one embodiment, the cancer to be treatedis multiple myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD44v6CAR, whereinthe cancer cells express CD44v6. In one embodiment, the cancer to betreated is cervical cancer, AML, or MM.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CEACAR, wherein thecancer cells express CEA. In one embodiment, the cancer to be treated isgastrointestinal cancer, or pancreatic cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an EPCAMCAR, whereinthe cancer cells express EPCAM. In one embodiment, the cancer to betreated is gastrointestinal cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a B7H3CAR, wherein thecancer cells express B7H3.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a KITCAR, wherein thecancer cells express KIT. In one embodiment, the cancer to be treated isgastrointestinal cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an IL-13Ra2CAR,wherein the cancer cells express IL-13Ra2. In one embodiment, the cancerto be treated is glioblastoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PRSS21CAR, whereinthe cancer cells express PRSS21. In one embodiment, the cancer to betreated is selected from ovarian, pancreatic, lung and breast cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD30CAR, wherein thecancer cells express CD30. In one embodiment, the cancer to be treatedis lymphomas, or leukemias.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GD3CAR, wherein thecancer cells express GD3. In one embodiment, the cancer to be treated ismelanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD171CAR, whereinthe cancer cells express CD171. In one embodiment, the cancer to betreated is neuroblastoma, ovarian cancer, melanoma, breast cancer,pancreatic cancer, colon cancers, or NSCLC (non-small cell lung cancer).

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an IL-11RaCAR, whereinthe cancer cells express IL-11Ra. In one embodiment, the cancer to betreated is osteosarcoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PSCACAR, wherein thecancer cells express PSCA. In one embodiment, the cancer to be treatedis prostate cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a VEGFR2CAR, whereinthe cancer cells express VEGFR2. In one embodiment, the cancer to betreated is a solid tumor.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LewisYCAR, whereinthe cancer cells express LewisY. In one embodiment, the cancer to betreated is ovarian cancer, or AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD24CAR, wherein thecancer cells express CD24. In one embodiment, the cancer to be treatedis pancreatic cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PDGFR-betaCAR,wherein the cancer cells express PDGFR-beta. In one embodiment, thecancer to be treated is breast cancer, prostate cancer, GIST(gastrointestinal stromal tumor), CML, DFSP (dermatofibrosarcomaprotuberans), or glioma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a SSEA-4CAR, whereinthe cancer cells express SSEA-4. In one embodiment, the cancer to betreated is glioblastoma, breast cancer, lung cancer, or stem cellcancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD20CAR, wherein thecancer cells express CD20. In one embodiment, the cancer to be treatedis B cell malignancies.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Folate receptoralphaCAR, wherein the cancer cells express folate receptor alpha.

In one embodiment, the cancer to be treated is ovarian cancer, NSCLC,endometrial cancer, renal cancer, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an ERBB2CAR, whereinthe cancer cells express ERBB2 (Her2/neu). In one embodiment, the cancerto be treated is breast cancer, gastric cancer, colorectal cancer, lungcancer, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MUC1CAR, wherein thecancer cells express MUC1. In one embodiment, the cancer to be treatedis breast cancer, lung cancer, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an EGFRCAR, whereinthe cancer cells express EGFR. In one embodiment, the cancer to betreated is glioblastoma, SCLC (small cell lung cancer), SCCHN (squamouscell carcinoma of the head and neck), NSCLC, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a NCAMCAR, wherein thecancer cells express NCAM. In one embodiment, the cancer to be treatedis neuroblastoma, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CAIXCAR, wherein thecancer cells express CAIX. In one embodiment, the cancer to be treatedis renal cancer, CRC, cervical cancer, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an EphA2CAR, whereinthe cancer cells express EphA2. In one embodiment, the cancer to betreated is GBM.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GD3CAR, wherein thecancer cells express GD3. In one embodiment, the cancer to be treated ismelanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Fucosyl GM1CAR,wherein the cancer cells express Fucosyl GM

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a sLeCAR, wherein thecancer cells express sLe. In one embodiment, the cancer to be treated isNSCLC, or AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GM3CAR, wherein thecancer cells express GM3.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TGS5CAR, wherein thecancer cells express TGS5.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a HMWMAACAR, whereinthe cancer cells express HMWMAA. In one embodiment, the cancer to betreated is melanoma, glioblastoma, or breast cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an o-acetyl-GD2CAR,wherein the cancer cells express o-acetyl-GD2. In one embodiment, thecancer to be treated is neuroblastoma, or melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD19CAR, wherein thecancer cells express CD19. In one embodiment, the cancer to be treatedis Follicular lymphoma, CLL, ALL, or myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TEM1/CD248CAR,wherein the cancer cells express TEM1/CD248. In one embodiment, thecancer to be treated is a solid tumor.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TEM7RCAR, whereinthe cancer cells express TEM7R. In one embodiment, the cancer to betreated is solid tumor.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CLDN6CAR, whereinthe cancer cells express CLDN6. In one embodiment, the cancer to betreated is ovarian cancer, lung cancer, or breast cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TSHRCAR, wherein thecancer cells express TSHR. In one embodiment, the cancer to be treatedis thyroid cancer, or multiple myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GPRC5DCAR, whereinthe cancer cells express GPRC5D. In one embodiment, the cancer to betreated is multiple myeloma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CXORF61CAR, whereinthe cancer cells express CXORF61.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD97CAR, wherein thecancer cells express CD97. In one embodiment, the cancer to be treatedis B cell malignancies, gastric cancer, pancreatic cancer, esophagealcancer, glioblastoma, breast cancer, or colorectal cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD179aCAR, whereinthe cancer cells express CD179a. In one embodiment, the cancer to betreated is B cell malignancies.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an ALK CAR, whereinthe cancer cells express ALK. In one embodiment, the cancer to betreated is NSCLC, ALCL (anaplastic large cell lymphoma), IMT(inflammatory myofibroblastic tumor), or neuroblastoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Polysialic acid CAR,wherein the cancer cells express Polysialic acid. In one embodiment, thecancer to be treated is small cell lung cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PLAC1CAR, whereinthe cancer cells express PLAC1. In one embodiment, the cancer to betreated is HCC (hepatocellular carcinoma).

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GloboHCAR, whereinthe cancer cells express GloboH. In one embodiment, the cancer to betreated is ovarian cancer, gastric cancer, prostate cancer, lung cancer,breast cancer, or pancreatic cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a NY-BR-1CAR, whereinthe cancer cells express NY-BR-1. In one embodiment, the cancer to betreated is breast cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a UPK2CAR, wherein thecancer cells express UPK2. In one embodiment, the cancer to be treatedis bladder cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a HAVCR1CAR, whereinthe cancer cells express HAVCR1. In one embodiment, the cancer to betreated is renal cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a ADRB3CAR, whereinthe cancer cells express ADRB3. In one embodiment, the cancer to betreated is Ewing sarcoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PANX3CAR, whereinthe cancer cells express PANX3. In one embodiment, the cancer to betreated is osteosarcoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GPR20CAR, whereinthe cancer cells express GPR20. In one embodiment, the cancer to betreated is GIST.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LY6KCAR, wherein thecancer cells express LY6K. In one embodiment, the cancer to be treatedis breast cancer, lung cancer, ovary cancer, or cervix cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a OR51E2CAR, whereinthe cancer cells express OR51E2. In one embodiment, the cancer to betreated is prostate cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TARPCAR, wherein thecancer cells express TARP. In one embodiment, the cancer to be treatedis prostate cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a WT1CAR, wherein thecancer cells express WT1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a NY-ESO-1CAR, whereinthe cancer cells express NY-ESO-1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LAGE-1a CAR, whereinthe cancer cells express LAGE-1a.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MAGE-A1CAR, whereinthe cancer cells express MAGE-A1. In one embodiment, the cancer to betreated is melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MAGE A1CAR, whereinthe cancer cells express MAGE A1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a ETV6-AML CAR,wherein the cancer cells express ETV6-AML.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a sperm protein 17CAR, wherein the cancer cells express sperm protein 17. In oneembodiment, the cancer to be treated is ovarian cancer, HCC, or NSCLC.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a XAGE1CAR, whereinthe cancer cells express XAGE1. In one embodiment, the cancer to betreated is Ewings, or rhabdo cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Tie 2 CAR, whereinthe cancer cells express Tie 2. In one embodiment, the cancer to betreated is a solid tumor.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MAD-CT-1CAR, whereinthe cancer cells express MAD-CT-1. In one embodiment, the cancer to betreated is prostate cancer, or melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MAD-CT-2CAR, whereinthe cancer cells express MAD-CT-2. In one embodiment, the cancer to betreated is prostate cancer, melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Fos-related antigen1 CAR, wherein the cancer cells express Fos-related antigen 1. In oneembodiment, the cancer to be treated is glioma, squamous cell cancer, orpancreatic cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a p53CAR, wherein thecancer cells express p53.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a prostein CAR,wherein the cancer cells express prostein.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a survivin andtelomerase CAR, wherein the cancer cells express survivin andtelomerase.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PCTA-1/Galectin 8CAR, wherein the cancer cells express PCTA-1/Galectin 8.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MelanA/MART1CAR,wherein the cancer cells express MelanA/MART1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Ras mutant CAR,wherein the cancer cells express Ras mutant.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a p53 mutant CAR,wherein the cancer cells express p53 mutant.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a hTERT CAR, whereinthe cancer cells express hTERT.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a sarcomatranslocation breakpoints CAR, wherein the cancer cells express sarcomatranslocation breakpoints. In one embodiment, the cancer to be treatedis sarcoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a ML-IAP CAR, whereinthe cancer cells express ML-IAP. In one embodiment, the cancer to betreated is melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an ERGCAR, wherein thecancer cells express ERG (TMPRSS2 ETS fusion gene).

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a NA17CAR, wherein thecancer cells express NA17. In one embodiment, the cancer to be treatedis melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PAX3CAR, wherein thecancer cells express PAX3. In one embodiment, the cancer to be treatedis alveolar rhabdomyosarcoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an androgen receptorCAR, wherein the cancer cells express androgen receptor. In oneembodiment, the cancer to be treated is metastatic prostate cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Cyclin B1CAR,wherein the cancer cells express Cyclin B1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a MYCNCAR, wherein thecancer cells express MYCN.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a RhoC CAR, whereinthe cancer cells express RhoC.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a TRP-2CAR, whereinthe cancer cells express TRP-2. In one embodiment, the cancer to betreated is melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CYP1B1CAR, whereinthe cancer cells express CYP1B1. In one embodiment, the cancer to betreated is breast cancer, colon cancer, lung cancer, esophagus cancer,skin cancer, lymph node cancer, brain cancer, or testis cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a BORIS CAR, whereinthe cancer cells express BORIS. In one embodiment, the cancer to betreated is lung cancer.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a SART3CAR, whereinthe cancer cells express SART3

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PAX5CAR, wherein thecancer cells express PAX5.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a OY-TES1CAR, whereinthe cancer cells express OY-TES1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LCK CAR, wherein thecancer cells express LCK.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a AKAP-4CAR, whereinthe cancer cells express AKAP-4.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a SSX2CAR, wherein thecancer cells express SSX2.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a RAGE-1CAR, whereinthe cancer cells express RAGE-1. In one embodiment, the cancer to betreated is RCC (renal cell cancer), or other solid tumors

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a human telomerasereverse transcriptase CAR, wherein the cancer cells express humantelomerase reverse transcriptase. In one embodiment, the cancer to betreated is solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a RU1CAR, wherein thecancer cells express RU1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a RU2CAR, wherein thecancer cells express RU2.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an intestinal carboxylesterase CAR, wherein the cancer cells express intestinal carboxylesterase. In one embodiment, the cancer to be treated is thyroid cancer,RCC, CRC (colorectal cancer), breast cancer, or other solid tumors.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Prostase CAR,wherein the cancer cells express Prostase.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a PAPCAR, wherein thecancer cells express PAP.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an IGF-I receptor CAR,wherein the cancer cells express IGF-I receptor.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a gp100 CAR, whereinthe cancer cells express gp100.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a bcr-abl CAR, whereinthe cancer cells express bcr-abl.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a tyrosinase CAR,wherein the cancer cells express tyrosinase.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a Fucosyl GM1CAR,wherein the cancer cells express Fucosyl GME

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a mut hsp70-2CAR,wherein the cancer cells express mut hsp70-2. In one embodiment, thecancer to be treated is melanoma.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD79a CAR, whereinthe cancer cells express CD79a.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD79b CAR, whereinthe cancer cells express CD79b.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD72 CAR, whereinthe cancer cells express CD72.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LAIR1 CAR, whereinthe cancer cells express LAIR1.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a FCAR CAR, whereinthe cancer cells express FCAR.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LILRA2 CAR, whereinthe cancer cells express LILRA2.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CD300LF CAR, whereinthe cancer cells express CD300LF.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a CLEC12A CAR, whereinthe cancer cells express CLEC12A.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a BST2 CAR, whereinthe cancer cells express BST2.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an EMR2 CAR, whereinthe cancer cells express EMR2.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a LY75 CAR, whereinthe cancer cells express LY75.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a GPC3 CAR, whereinthe cancer cells express GPC3.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express a FCRL5 CAR, whereinthe cancer cells express FCRL5.

In one aspect, the present invention provides methods of treating cancerby providing to the subject in need thereof immune effector cells (e.g.,T cells, NK cells) that are engineered to express an IGLL1 CAR, whereinthe cancer cells express IGLL1.

In one aspect, the present invention relates to treatment of a subjectin vivo using an PD1 CAR such that growth of cancerous tumors isinhibited. A PD1 CAR may be used alone to inhibit the growth ofcancerous tumors. Alternatively, PD1 CAR may be used in conjunction withother CARs, immunogenic agents, standard cancer treatments, or otherantibodies. In one embodiment, the subject is treated with a PD1 CAR andan XCAR described herein. In an embodiment, a PD1 CAR is used inconjunction with another CAR, e.g., a CAR described herein, and a kinaseinhibitor, e.g., a kinase inhibitor described herein.

In another aspect, a method of treating a subject, e.g., reducing orameliorating, a hyperproliferative condition or disorder (e.g., acancer), e.g., solid tumor, a soft tissue tumor, or a metastatic lesion,in a subject is provided. As used herein, the term “cancer” is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. Examplesof solid tumors include malignancies, e.g., sarcomas, adenocarcinomas,and carcinomas, of the various organ systems, such as those affectingliver, lung, breast, lymphoid, gastrointestinal (e.g., colon),genitourinary tract (e.g., renal, urothelial cells), prostate andpharynx. Adenocarcinomas include malignancies such as most coloncancers, rectal cancer, renal-cell carcinoma, liver cancer, non-smallcell carcinoma of the lung, cancer of the small intestine and cancer ofthe esophagus. In one embodiment, the cancer is a melanoma, e.g., anadvanced stage melanoma. Metastatic lesions of the aforementionedcancers can also be treated or prevented using the methods andcompositions of the invention. Examples of other cancers that can betreated include bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease,non-Hodgkin lymphoma, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumorangiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-celllymphoma, environmentally induced cancers including those induced byasbestos, and combinations of said cancers. Treatment of metastaticcancers, e.g., metastatic cancers that express PD-L1 (Iwai et al. (2005)Int. Immunol. 17:133-144) can be effected using the antibody moleculesdescribed herein.

Exemplary cancers whose growth can be inhibited include cancerstypically responsive to immunotherapy. Non-limiting examples of cancersfor treatment include melanoma (e.g., metastatic malignant melanoma),renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), breast cancer, colon cancer andlung cancer (e.g. non-small cell lung cancer). Additionally, refractoryor recurrent malignancies can be treated using the molecules describedherein.

In one aspect, the invention pertains to a vector comprising a CARoperably linked to promoter for expression in mammalian immune effectorcells (e.g., T cells, NK cells). In one aspect, the invention provides arecombinant immune effector cell expressing a CAR of the presentinvention for use in treating cancer expressing a cancer associateantigen as described herein. In one aspect, CAR-expressing cells of theinvention is capable of contacting a tumor cell with at least one cancerassociated antigen expressed on its surface such that the CAR-expressingcell targets the cancer cell and growth of the cancer is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a cancer, comprising contacting the cancer cell with a CAR-expressingcell of the present invention such that the CART is activated inresponse to the antigen and targets the cancer cell, wherein the growthof the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subjectCAR-expressing cell of the present invention such that the cancer istreated in the subject. In one aspect, the cancer associated withexpression of a cancer associate antigen as described herein is ahematological cancer. In one aspect, the hematological cancer is aleukemia or a lymphoma. In one aspect, a cancer associated withexpression of a cancer associate antigen as described herein includescancers and malignancies including, but not limited to, e.g., one ormore acute leukemias including but not limited to, e.g., B-cell acuteLymphoid Leukemia (“BALL”), T-cell acute Lymphoid Leukemia (“TALL”),acute lymphoid leukemia (ALL); one or more chronic leukemias includingbut not limited to, e.g., chronic myelogenous leukemia (CML), ChronicLymphoid Leukemia (CLL). Additional cancers or hematologic conditionsassociated with expression of a cancer associate antigen as describedherein include, but are not limited to, e.g., B cell prolymphocyticleukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'slymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma,Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and the like. Further a disease associated with a cancer associateantigen as described herein expression include, but not limited to,e.g., atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases associated with expression of acancer associate antigen as described herein.

In some embodiments, a cancer that can be treated with CAR-expressingcell of the present invention is multiple myeloma. Multiple myeloma is acancer of the blood, characterized by accumulation of a plasma cellclone in the bone marrow. Current therapies for multiple myelomainclude, but are not limited to, treatment with lenalidomide, which isan analog of thalidomide. Lenalidomide has activities which includeanti-tumor activity, angiogenesis inhibition, and immunomodulation.Generally, myeloma cells are thought to be negative for a cancerassociate antigen as described herein expression by flow cytometry.Thus, in some embodiments, a CD19 CAR, e.g., as described herein, may beused to target myeloma cells. In some embodiments, cars of the presentinvention therapy can be used in combination with one or more additionaltherapies, e.g., lenalidomide treatment.

The invention includes a type of cellular therapy where immune effectorcells (e.g., T cells, NK cells) are genetically modified to express achimeric antigen receptor (CAR) and the CAR-expressing T cell or NK cellis infused to a recipient in need thereof. The infused cell is able tokill tumor cells in the recipient. Unlike antibody therapies,CAR-modified immune effector cells (e.g., T cells, NK cells) are able toreplicate in vivo resulting in long-term persistence that can lead tosustained tumor control. In various aspects, the immune effector cells(e.g., T cells, NK cells) administered to the patient, or their progeny,persist in the patient for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the T cell or NK cell to the patient.

The invention also includes a type of cellular therapy where immuneeffector cells (e.g., T cells, NK cells) are modified, e.g., by in vitrotranscribed RNA, to transiently express a chimeric antigen receptor(CAR) and the CAR T cell or NK cell is infused to a recipient in needthereof. The infused cell is able to kill tumor cells in the recipient.Thus, in various aspects, the immune effector cells (e.g., T cells, NKcells) administered to the patient, is present for less than one month,e.g., three weeks, two weeks, one week, after administration of the Tcell or NK cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified immune effector cells(e.g., T cells, NK cells) may be an active or a passive immune response,or alternatively may be due to a direct vs indirect immune response. Inone aspect, the CAR transduced immune effector cells (e.g., T cells, NKcells) exhibit specific proinflammatory cytokine secretion and potentcytolytic activity in response to human cancer cells expressing the acancer associate antigen as described herein, resist soluble a cancerassociate antigen as described herein inhibition, mediate bystanderkilling and mediate regression of an established human tumor. Forexample, antigen-less tumor cells within a heterogeneous field of acancer associate antigen as described herein-expressing tumor may besusceptible to indirect destruction by a cancer associate antigen asdescribed herein-redirected immune effector cells (e.g., T cells, NKcells) that has previously reacted against adjacent antigen-positivecancer cells.

In one aspect, the fully-human CAR-modified immune effector cells (e.g.,T cells, NK cells) of the invention may be a type of vaccine for ex vivoimmunization and/or in vivo therapy in a mammal. In one aspect, themammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of immune effector cells(e.g., T cells, NK cells) comprises: (1) collecting CD34+ hematopoieticstem and progenitor cells from a mammal from peripheral blood harvest orbone marrow explants; and (2) expanding such cells ex vivo. In additionto the cellular growth factors described in U.S. Pat. No. 5,199,942,other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be usedfor culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modifiedimmune effector cells (e.g., T cells, NK cells) of the invention areused in the treatment of diseases, disorders and conditions associatedwith expression of a cancer associate antigen as described herein. Incertain aspects, the cells of the invention are used in the treatment ofpatients at risk for developing diseases, disorders and conditionsassociated with expression of a cancer associate antigen as describedherein. Thus, the present invention provides methods for the treatmentor prevention of diseases, disorders and conditions associated withexpression of a cancer associate antigen as described herein comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified immune effector cells (e.g., T cells, NKcells) of the invention.

In one aspect the CAR-expressing cells of the inventions may be used totreat a proliferative disease such as a cancer or malignancy or is aprecancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia. Further a disease associated with a cancerassociate antigen as described herein expression include, but notlimited to, e.g., atypical and/or non-classical cancers, malignancies,precancerous conditions or proliferative diseases expressing a cancerassociated antigen as described herein. Non-cancer related indicationsassociated with expression of a cancer associate antigen as describedherein include, but are not limited to, e.g., autoimmune disease, (e.g.,lupus), inflammatory disorders (allergy and asthma) and transplantation.

The CAR-modified immune effector cells (e.g., T cells, NK cells) of thepresent invention may be administered either alone, or as apharmaceutical composition in combination with diluents and/or withother components such as IL-2 or other cytokines or cell populations.

Hematologic Cancer

Hematological cancer conditions are the types of cancer such asleukemia, lymphoma, and malignant lymphoproliferative conditions thataffect blood, bone marrow and the lymphatic system.

Leukemia can be classified as acute leukemia and chronic leukemia. Acuteleukemia can be further classified as acute myelogenous leukemia (AML)and acute lymphoid leukemia (ALL). Chronic leukemia includes chronicmyelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Otherrelated conditions include myelodysplastic syndromes (MDS, formerlyknown as “preleukemia”) which are a diverse collection of hematologicalconditions united by ineffective production (or dysplasia) of myeloidblood cells and risk of transformation to AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes.Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

The present invention provides for compositions and methods for treatingcancer. In one aspect, the cancer is a hematologic cancer including butis not limited to hematological cancer is a leukemia or a lymphoma. Inone aspect, the CAR-expressing cells of the invention may be used totreat cancers and malignancies such as, but not limited to, e.g., acuteleukemias including but not limited to, e.g., B-cell acute lymphoidleukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acutelymphoid leukemia (ALL); one or more chronic leukemias including but notlimited to, e.g., chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL); additional hematologic cancers orhematologic conditions including, but not limited to, e.g., B cellprolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma,Hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and the like. Further a disease associated with a cancer associateantigen as described herein expression includes, but not limited to,e.g., atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases expressing a cancer associateantigen as described herein.

The present invention also provides methods for inhibiting theproliferation or reducing a cancer associated antigen as describedherein-expressing cell population, the methods comprising contacting apopulation of cells comprising a cancer associated antigen as describedherein-expressing cell with a CAR-expressing T cell or NK cell of theinvention that binds to the a cancer associate antigen as describedherein-expressing cell. In a specific aspect, the present inventionprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing a cancer associated antigen asdescribed herein, the methods comprising contacting a cancer associateantigen as described herein-expressing cancer cell population with aCAR-expressing T cell or NK cell of the invention that binds to a cancerassociated antigen as described herein-expressing cell. In one aspect,the present invention provides methods for inhibiting the proliferationor reducing the population of cancer cells expressing a cancerassociated antigen as described herein, the methods comprisingcontacting a cancer associated antigen as described herein-expressingcancer cell population with a CAR-expressing T cell or NK cell of theinvention that binds to a cancer associated antigen as describedherein-expressing cell. In certain aspects, a CAR-expressing T cell orNK cell of the invention reduces the quantity, number, amount orpercentage of cells and/or cancer cells by at least 25%, at least 30%,at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, atleast 95%, or at least 99% in a subject with or animal model for myeloidleukemia or another cancer associated with a cancer associated antigenas described herein-expressing cells relative to a negative control. Inone aspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with a cancer associated antigen asdescribed herein-expressing cells (e.g., a hematologic cancer oratypical cancer expressing a cancer associated antigen as describedherein), the methods comprising administering to a subject in need a CART cell or NK cell of the invention that binds to a cancer associatedantigen as described herein-expressing cell. In one aspect, the subjectis a human Non-limiting examples of disorders associated with a cancerassociated antigen as described herein-expressing cells includeautoimmune disorders (such as lupus), inflammatory disorders (such asallergies and asthma) and cancers (such as hematological cancers oratypical cancers expressing a cancer associated antigen as describedherein).

The present invention also provides methods for preventing, treatingand/or managing a disease associated with a cancer associated antigen asdescribed herein-expressing cells, the methods comprising administeringto a subject in need a CAR T cell or NK cell of the invention that bindsto a cancer associated antigen as described herein-expressing cell. Inone aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with a cancer associated antigen as describedherein-expressing cells, the methods comprising administering to asubject in need thereof a CAR T cell or NK cell of the invention thatbinds to a cancer associated antigen as described herein-expressingcell. In one aspect, the methods comprise administering to the subjectin need thereof an effective amount of a CAR-expressing T cell or NKcell described herein that binds to a cancer associated antigen asdescribed herein-expressing cell in combination with an effective amountof another therapy.

Combination Therapies

A CAR-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CAR therapy canbe administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and the additionalagent (e.g., second or third agent), or all, can be administered in anamount or dose that is higher, lower or the same than the amount ordosage of each agent used individually, e.g., as a monotherapy. Incertain embodiments, the administered amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all, islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)than the amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as n, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation.peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include an anthracycline (e.g., doxorubicin (e.g., liposomaldoxorubicin)). a vinca alkaloid (e.g., vinblastine, vincristine,vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide,decarbazine, melphalan, ifosfamide, temozolomide), an immune cellantibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab,tositumomab, brentuximab), an antimetabolite (including, e.g., folicacid antagonists, pyrimidine analogs, purine analogs and adenosinedeaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFRglucocorticoid induced TNFR related protein (GITR) agonist, a proteasomeinhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), animmunomodulator such as thalidomide or a thalidomide derivative (e.g.,lenalidomide).

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

Exemplary alkylating agents include, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®,Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracilnitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®,Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®,Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide(Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman(Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®),triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa(Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®),lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine(DTIC-Dome®). Additional exemplary alkylating agents include, withoutlimitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® andTemodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®);Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard,Alkeran®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan(Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (alsoknown as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® andPlatinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® andNeosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine(HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine(Matulane®); Mechlorethamine (also known as nitrogen mustard, mustineand mechloroethamine hydrochloride, Mustargen®); Streptozocin(Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA,Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®,Revimmune®); and Bendamustine HCl (Treanda®).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with fludarabine, cyclophosphamide, and/orrituximab. In embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with fludarabine,cyclophosphamide, and rituximab (FCR). In embodiments, the subject hasCLL. For example, the subject has a deletion in the short arm ofchromosome 17 (del(17p), e.g., in a leukemic cell). In other examples,the subject does not have a del(17p). In embodiments, the subjectcomprises a leukemic cell comprising a mutation in the immunoglobulinheavy-chain variable-region (IgV_(H)) gene. In other embodiments, thesubject does not comprise a leukemic cell comprising a mutation in theimmunoglobulin heavy-chain variable-region (IgV_(H)) gene. Inembodiments, the fludarabine is administered at a dosage of about 10-50mg/m² (e.g., about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or45-50 mg/m²), e.g., intravenously. In embodiments, the cyclophosphamideis administered at a dosage of about 200-300 mg/m² (e.g., about 200-225,225-250, 250-275, or 275-300 mg/m²), e.g., intravenously. Inembodiments, the rituximab is administered at a dosage of about 400-600mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m²), e.g.,intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with bendamustine and rituximab. Inembodiments, the subject has CLL. For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the bendamustine isadministered at a dosage of about 70-110 mg/m2 (e.g., 70-80, 80-90,90-100, or 100-110 mg/m2), e.g., intravenously. In embodiments, therituximab is administered at a dosage of about 400-600 mg/m2 (e.g.,400-450, 450-500, 500-550, or 550-600 mg/m²), e.g., intravenously.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and/or a corticosteroid (e.g., prednisone).In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab, cyclophosphamide,doxorubicine, vincristine, and prednisone (R-CHOP). In embodiments, thesubject has diffuse large B-cell lymphoma (DLBCL). In embodiments, thesubject has nonbulky limited-stage DLBCL (e.g., comprises a tumor havinga size/diameter of less than 7 cm). In embodiments, the subject istreated with radiation in combination with the R-CHOP. For example, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP), followed by radiation. In some cases, thesubject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5,or 6 cycles of R-CHOP) following radiation.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with etoposide, prednisone, vincristine,cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, aCAR-expressing cell described herein is administered to a subject incombination with etoposide, prednisone, vincristine, cyclophosphamide,doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressingcell described herein is administered to a subject in combination withdose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a Bcell lymphoma, e.g., a Myc-rearranged aggressive B cell lymphoma.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with rituximab and/or lenalidomide.Lenalidomide ((RS)-3-(4-Amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is animmunomodulator. In embodiments, a CAR-expressing cell described hereinis administered to a subject in combination with rituximab andlenalidomide. In embodiments, the subject has follicular lymphoma (FL)or mantle cell lymphoma (MCL). In embodiments, the subject has FL andhas not previously been treated with a cancer therapy. In embodiments,lenalidomide is administered at a dosage of about 10-20 mg (e.g., 10-15or 15-20 mg), e.g., daily. In embodiments, rituximab is administered ata dosage of about 350-550 mg/m² (e.g., 350-375, 375-400, 400-425,425-450, 450-475, or 475-500 mg/m²), e.g., intravenously.

Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus(formally known as deferolimus,(1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQID NO: 112), inner salt (SF1126, CAS 936487-67-1), and XL765.

Exemplary immunomodulators include, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon γ, CAS951209-71-5, available from IRX Therapeutics).

Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone(DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin;ravidomycin; and desacetylravidomycin.

Exemplary vinca alkaloids include, e.g., vinorelbine tartrate(Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®));vinblastine (also known as vinblastine sulfate, vincaleukoblastine andVLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

Exemplary proteosome inhibitors include bortezomib (Velcade®);carfilzomib (PX-171-007,(S)-4-Methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide);marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib(CEP-18770); andO-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide(ONX-0912).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab. Brentuximab is anantibody-drug conjugate of anti-CD30 antibody and monomethyl auristatinE. In embodiments, the subject has Hodgkin's lymphoma (HL), e.g.,relapsed or refractory HL. In embodiments, the subject comprisesCD30+HL. In embodiments, the subject has undergone an autologous stemcell transplant (ASCT). In embodiments, the subject has not undergone anASCT. In embodiments, brentuximab is administered at a dosage of about1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g.,intravenously, e.g., every 3 weeks.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with brentuximab and dacarbazine or incombination with brentuximab and bendamustine. Dacarbazine is analkylating agent with a chemical name of5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is analkylating agent with a chemical name of4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid.In embodiments, the subject has Hodgkin's lymphoma (HL). In embodiments,the subject has not previously been treated with a cancer therapy. Inembodiments, the subject is at least 60 years of age, e.g., 60, 65, 70,75, 80, 85, or older. In embodiments, dacarbazine is administered at adosage of about 300-450 mg/m² (e.g., about 300-325, 325-350, 350-375,375-400, 400-425, or 425-450 mg/m²), e.g., intravenously. Inembodiments, bendamustine is administered at a dosage of about 75-125mg/m2 (e.g., 75-100 or 100-125 mg/m², e.g., about 90 mg/m²), e.g.,intravenously. In embodiments, brentuximab is administered at a dosageof about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg),e.g., intravenously, e.g., every 3 weeks.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a CD20 inhibitor, e.g., ananti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific antibody) ora fragment thereof. Exemplary anti-CD20 antibodies include but are notlimited to rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab,TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921(Genentech). See, e.g., Lim et al. Haematologica. 95.1 (2010):135-43.

In some embodiments, the anti-CD20 antibody comprises rituximab.Rituximab is a chimeric mouse/human monoclonal antibody IgG1 kappa thatbinds to CD20 and causes cytolysis of a CD20 expressing cell, e.g., asdescribed inwww.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s53111bl.pdf. Inembodiments, a CAR-expressing cell described herein is administered to asubject in combination with rituximab. In embodiments, the subject hasCLL or SLL.

In some embodiments, rituximab is administered intravenously, e.g., asan intravenous infusion. For example, each infusion provides about500-2000 mg (e.g., about 500-550, 550-600, 600-650, 650-700, 700-750,750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200,1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,1800-1900, or 1900-2000 mg) of rituximab. In some embodiments, rituximabis administered at a dose of 150 mg/m² to 750 mg/m², e.g., about 150-175mg/m², 175-200 mg/m², 200-225 mg/m², 225-250 mg/m², 250-300 mg/m²,300-325 mg/m², 325-350 mg/m², 350-375 mg/m², 375-400 mg/m², 400-425mg/m², 425-450 mg/m², 450-475 mg/m², 475-500 mg/m², 500-525 mg/m²,525-550 mg/m², 550-575 mg/m², 575-600 mg/m², 600-625 mg/m², 625-650mg/m², 650-675 mg/m², or 675-700 mg/m², where m² indicates the bodysurface area of the subject. In some embodiments, rituximab isadministered at a dosing interval of at least 4 days, e.g., 4, 7, 14,21, 28, 35 days, or more. For example, rituximab is administered at adosing interval of at least 0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8weeks, or more. In some embodiments, rituximab is administered at a doseand dosing interval described herein for a period of time, e.g., atleast 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 weeks, or greater. For example, rituximab isadministered at a dose and dosing interval described herein for a totalof at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or more doses per treatment cycle).

In some embodiments, the anti-CD20 antibody comprises ofatumumab.Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody with amolecular weight of approximately 149 kDa. For example, ofatumumab isgenerated using transgenic mouse and hybridoma technology and isexpressed and purified from a recombinant murine cell line (NS0). See,e.g., www.accessdata.fda.gov/drugsatfda_docs/label/2009/1253261bl.pdf;and Clinical Trial Identifier number NCT01363128, NCT01515176,NCT01626352, and NCT01397591. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withofatumumab. In embodiments, the subject has CLL or SLL.

In some embodiments, ofatumumab is administered as an intravenousinfusion. For example, each infusion provides about 150-3000 mg (e.g.,about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800,1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000 mg)of ofatumumab. In embodiments, ofatumumab is administered at a startingdosage of about 300 mg, followed by 2000 mg, e.g., for about 11 doses,e.g., for 24 weeks. In some embodiments, ofatumumab is administered at adosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, ormore. For example, ofatumumab is administered at a dosing interval of atleast 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28,20, 22, 24, 26, 28, 30 weeks, or more. In some embodiments, ofatumumabis administered at a dose and dosing interval described herein for aperiod of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50,60 weeks or greater, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months orgreater, or 1, 2, 3, 4, 5 years or greater. For example, ofatumumab isadministered at a dose and dosing interval described herein for a totalof at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or more doses per treatmentcycle).

In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumabis a humanized anti-CD20 monoclonal antibody, e.g., as described inClinical Trials Identifier Nos. NCT00077870, NCT01412333, NCT00779220,NCT00673920, NCT01194570, and Kappos et al. Lancet. 19.378(2011):1779-87.

In some cases, the anti-CD20 antibody comprises veltuzumab. Veltuzumabis a humanized monoclonal antibody against CD20. See, e.g., ClinicalTrial Identifier No. NCT00547066, NCT00546793, NCT01101581, andGoldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

In some cases, the anti-CD20 antibody comprises GA101. GA101 (alsocalled obinutuzumab or RO5072759) is a humanized and glyco-engineeredanti-CD20 monoclonal antibody. See, e.g., Robak. Curr. Opin. Investig.Drugs. 10.6 (2009):588-96; Clinical Trial Identifier Numbers:NCT01995669, NCT01889797, NCT02229422, and NCT01414205; andwww.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s0001bl.pdf.

In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (alsocalled LY2469298 or ocaratuzumab) is a humanized IgG1 monoclonalantibody against CD20 with increased affinity for the FcγRIIIa receptorand an enhanced antibody dependent cellular cytotoxicity (ADCC) activitycompared with rituximab. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res. 18.5(2012):1395-403.

In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 isa humanized anti-CD20 monoclonal antibody engineered to have betterbinding to FcγRIIIa and enhanced ADCC compared with rituximab. See,e.g., Robak et al. BioDrugs 25.1 (2011):13-25; and Casulo et al. ClinImmunol. 154.1 (2014):37-46; and Clinical Trial Identifier No.NCT00452127.

In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is ananti-CD20 fusion protein derived from domains of an antibody againstCD20. TRU-015 is smaller than monoclonal antibodies, but retainsFc-mediated effector functions. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variablefragment (scFv) linked to human IgG1 hinge, CH2, and CH3 domains butlacks CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein isconjugated or otherwise bound to a therapeutic agent, e.g., achemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylaseinhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic,tyrosine kinase inhibitor, alkylating agent, anti-microtubule oranti-mitotic agent), anti-allergic agent, anti-nausea agent (oranti-emetic), pain reliever, or cytoprotective agent described herein.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a B-cell lymphoma 2 (BCL-2) inhibitor(e.g., venetoclax, also called ABT-199 or GDC-0199;) and/or rituximab.In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with venetoclax and rituximab. Venetoclax isa small molecule that inhibits the anti-apoptotic protein, BCL-2. Thestructure of venetoclax(4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy. In embodiments, venetoclax is administered at a dosageof about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300,300-400, 400-500, or 500-600 mg), e.g., daily. In embodiments, rituximabis administered at a dosage of about 350-550 mg/m2 (e.g., 350-375,375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2), e.g.,intravenously, e.g., monthly

In an embodiment, cells expressing a CAR described herein areadministered to a subject in combination with a molecule that decreasesthe Treg cell population. Methods that decrease the number of (e.g.,deplete) Treg cells are known in the art and include, e.g., CD25depletion, cyclophosphamide administration, modulating GITR function.Without wishing to be bound by theory, it is believed that reducing thenumber of Treg cells in a subject prior to apheresis or prior toadministration of a CAR-expressing cell described herein reduces thenumber of unwanted immune cells (e.g., Tregs) in the tumormicroenvironment and reduces the subject's risk of relapse. In oneembodiment, cells expressing a CAR described herein are administered toa subject in combination with a molecule targeting GITR and/ormodulating GITR functions, such as a GITR agonist and/or a GITR antibodythat depletes regulatory T cells (Tregs). In embodiments, cellsexpressing a CAR described herein are administered to a subject incombination with cyclophosphamide. In one embodiment, the GITR bindingmolecules and/or molecules modulating GITR functions (e.g., GITR agonistand/or Treg depleting GITR antibodies) are administered prior toadministration of the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In embodiments, cyclophosphamide is administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In embodiments,cyclophosphamide and an anti-GITR antibody are administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In oneembodiment, the subject has cancer (e.g., a solid cancer or ahematological cancer such as ALL or CLL). In an embodiment, the subjecthas CLL. In embodiments, the subject has ALL. In embodiments, thesubject has a solid cancer, e.g., a solid cancer described herein.Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.:1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat.No. 8,591,886, European Patent No.: EP 1866339, PCT Publication No.: WO2011/028683, PCT Publication No.: WO 2013/039954, PCT Publication No.:WO2005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.:WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO2001/03720, PCT Publication No.: WO99/20758, PCT Publication No.:WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.7,618,632, and PCT Publication No.: WO 2011/051726.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with an mTOR inhibitor, e.g.,an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.In one embodiment, the mTOR inhibitor is administered prior to theCAR-expressing cell. For example, in one embodiment, the mTOR inhibitorcan be administered prior to apheresis of the cells. In one embodiment,the subject has CLL.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a GITR agonist, e.g., aGITR agonist described herein. In one embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In one embodiment, the subject has CLL.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a kinase inhibitor. In one embodiment, the kinaseinhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein,e.g., a CD4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. Inone embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitordescribed herein, such as, e.g., PF-04695102.

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selectedfrom aloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g.,palbociclib (PD0332991), and the palbociclib is administered at a doseof about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28 daycycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib areadministered.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a cyclin-dependent kinase (CDK) 4 or 6inhibitor, e.g., a CDK4 inhibitor or a CDK6 inhibitor described herein.In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a CDK4/6 inhibitor (e.g., an inhibitorthat targets both CDK4 and CDK6), e.g., a CDK4/6 inhibitor describedherein. In an embodiment, the subject has MCL. MCL is an aggressivecancer that is poorly responsive to currently available therapies, i.e.,essentially incurable. In many cases of MCL, cyclin D1 (a regulator ofCDK4/6) is expressed (e.g., due to chromosomal translocation involvingimmunoglobulin and Cyclin D1 genes) in MCL cells. Thus, without beingbound by theory, it is thought that MCL cells are highly sensitive toCDK4/6 inhibition with high specificity (i.e., minimal effect on normalimmune cells). CDK4/6 inhibitors alone have had some efficacy intreating MCL, but have only achieved partial remission with a highrelapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also calledribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that administration of aCAR-expressing cell described herein with a CDK4/6 inhibitor (e.g.,LEE011 or other CDK4/6 inhibitor described herein) can achieve higherresponsiveness, e.g., with higher remission rates and/or lower relapserates, e.g., compared to a CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected fromibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, theBTK inhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK), and is selected from GDC-0834;RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; andLFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib (PCI-32765). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with a BTK inhibitor(e.g., ibrutinib). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with ibrutinib (alsocalled PCI-32765). The structure of ibrutinib(1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one) is shown below.

In embodiments, the subject has CLL, mantle cell lymphoma (MCL), orsmall lymphocytic lymphoma (SLL). For example, the subject has adeletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject has relapsed CLL or SLL, e.g., the subjecthas previously been administered a cancer therapy (e.g., previously beenadministered one, two, three, or four prior cancer therapies). Inembodiments, the subject has refractory CLL or SLL. In otherembodiments, the subject has follicular lymphoma, e.g., relapse orrefractory follicular lymphoma. In some embodiments, ibrutinib isadministered at a dosage of about 300-600 mg/day (e.g., about 300-350,350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420mg/day or about 560 mg/day), e.g., orally. In embodiments, the ibrutinibis administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g.,daily for 21 day cycle, or daily for 28 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib areadministered. Without being bound by theory, it is thought that theaddition of ibrutinib enhances the T cell proliferative response and mayshift T cells from a T-helper-2 (Th2) to T-helper-1 (Th1) phenotype. Th1and Th2 are phenotypes of helper T cells, with Th1 versus Th2 directingdifferent immune response pathways. A Th1 phenotype is associated withproinflammatory responses, e.g., for killing cells, such asintracellular pathogens/viruses or cancerous cells, or perpetuatingautoimmune responses. A Th2 phenotype is associated with eosinophilaccumulation and anti-inflammatory responses.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selectedfrom temsirolimus; ridaforolimus(1R,2R,4S)-4-[*2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-(SEQ ID NO: 112), inner salt (SF1126); and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a periodof time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofrapamycin are administered. In one embodiment, the kinase inhibitor isan mTOR inhibitor, e.g., everolimus and the everolimus is administeredat a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for aperiod of time, e.g., daily for 28 day cycle. In one embodiment, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus areadministered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selectedfrom CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a phosphoinositide 3-kinase (PI3K)inhibitor (e.g., a PI3K inhibitor described herein, e.g., idelalisib orduvelisib) and/or rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withidelalisib and rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withduvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101;Gilead) is a small molecule that blocks the delta isoform of PI3K. Thestructure of idelalisib (5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is shown below.

Duvelisib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) isa small molecule that blocks PI3K-δ,γ. The structure of duvelisib(8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone)is shown below.

In embodiments, the subject has CLL. In embodiments, the subject hasrelapsed CLL, e.g., the subject has previously been administered acancer therapy (e.g., previously been administered an anti-CD20 antibodyor previously been administered ibrutinib). For example, the subject hasa deletion in the short arm of chromosome 17 (del(17p), e.g., in aleukemic cell). In other examples, the subject does not have a del(17p).In embodiments, the subject comprises a leukemic cell comprising amutation in the immunoglobulin heavy-chain variable-region (IgV_(H))gene. In other embodiments, the subject does not comprise a leukemiccell comprising a mutation in the immunoglobulin heavy-chainvariable-region (IgV_(H)) gene. In embodiments, the subject has adeletion in the long arm of chromosome 11 (del(11q)). In otherembodiments, the subject does not have a del(11q). In embodiments,idelalisib is administered at a dosage of about 100-400 mg (e.g.,100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300,325-350, 350-375, or 375-400 mg), e.g., BID. In embodiments, duvelisibis administered at a dosage of about 15-100 mg (e.g., about 15-25,25-50, 50-75, or 75-100 mg), e.g., twice a day. In embodiments,rituximab is administered at a dosage of about 350-550 mg/m² (e.g.,350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m²), e.g.,intravenously.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol3-kinase (PI3K) and mTOR inhibitor selected from2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-c]pyrimidin-7(8H)-one(PF-04691502);N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N′-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea(PF-05212384, PKI-587);2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile(BEZ-235); apitolisib (GDC-0980, RG7422);2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide(GSK2126458);8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-oneMaleic acid (NVP-BGT226);3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol(PI-103);5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(VS-5584, SB2343); andN-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide(XL765).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an anaplastic lymphoma kinase (ALK)inhibitor. Exemplary ALK kinases include but are not limited tocrizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai),brigatinib (also called AP26113; Ariad), entrectinib (Ignyta),PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, e.g., Clinical TrialIdentifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). Insome embodiments, the subject has a solid cancer, e.g., a solid cancerdescribed herein, e.g., lung cancer.

The chemical name of crizotinib is3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine.The chemical name of ceritinib is5-Chloro-N²-[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N⁴-[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine.The chemical name of alectinib is9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile.The chemical name of brigatinib is5-Chloro-N²-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N⁴-[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine.The chemical name of entrectinib isN-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide.The chemical name of PF-06463922 is(10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile.The chemical structure of CEP-37440 is(S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide.The chemical name of X-396 is(R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl)pyridazine-3-carboxamide.

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer etal., Curr. Opin. Immun 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMPATH. In one aspect, the cell compositionsof the present invention are administered following B-cell ablativetherapy such as agents that react with CD20, e.g., Rituxan. For example,in one embodiment, subjects may undergo standard treatment with highdose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with an indoleamine 2,3-dioxygenase (IDO)inhibitor. IDO is an enzyme that catalyzes the degradation of the aminoacid, L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g.,prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, andlung cancer. pDCs, macrophages, and dendritic cells (DCs) can expressIDO. Without being bound by theory, it is thought that a decrease inL-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressivemilieu by inducing T-cell anergy and apoptosis. Thus, without beingbound by theory, it is thought that an IDO inhibitor can enhance theefficacy of a CAR-expressing cell described herein, e.g., by decreasingthe suppression or death of a CAR-expressing immune cell. Inembodiments, the subject has a solid tumor, e.g., a solid tumordescribed herein, e.g., prostatic, colorectal, pancreatic, cervical,gastric, ovarian, head, or lung cancer. Exemplary inhibitors of IDOinclude but are not limited to 1-methyl-tryptophan, indoximod (NewLinkGenetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216;NCT01792050), and INCB024360 (Incyte Corp.) (see, e.g., Clinical TrialIdentifier Nos. NCT01604889; NCT01685255).

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a modulator of myeloid-derivedsuppressor cells (MDSCs). MDSCs accumulate in the periphery and at thetumor site of many solid tumors. These cells suppress T cell responses,thereby hindering the efficacy of CAR-expressing cell therapy. Withoutbeing bound by theory, it is thought that administration of a MDSCmodulator enhances the efficacy of a CAR-expressing cell describedherein. In an embodiment, the subject has a solid tumor, e.g., a solidtumor described herein, e.g., glioblastoma. Exemplary modulators ofMDSCs include but are not limited to MCS110 and BLZ945. MCS110 is amonoclonal antibody (mAb) against macrophage colony-stimulating factor(M-CSF). See, e.g., Clinical Trial Identifier No. NCT00757757. BLZ945 isa small molecule inhibitor of colony stimulating factor 1 receptor(CSF1R). See, e.g., Pyonteck et al. Nat. Med. 19(2013):1264-72. Thestructure of BLZ945 is shown below.

In embodiments, a CAR-expressing cell described herein is administeredto a subject in combination with a CD19 CART cell (e.g., CTL019, e.g.,as described in WO2012/079000, incorporated herein by reference). Inembodiments, the subject has a CD19+ lymphoma, e.g., a CD19+Non-Hodgkin's Lymphoma (NHL), a CD19+FL, or a CD19+ DLBCL. Inembodiments, the subject has a relapsed or refractory CD19+ lymphoma. Inembodiments, a lymphodepleting chemotherapy is administered to thesubject prior to, concurrently with, or after administration (e.g.,infusion) of CD19 CART cells. In an example, the lymphodepletingchemotherapy is administered to the subject prior to administration ofCD19 CART cells. For example, the lymphodepleting chemotherapy ends 1-4days (e.g., 1, 2, 3, or 4 days) prior to CD19 CART cell infusion. Inembodiments, multiple doses of CD19 CART cells are administered, e.g.,as described herein. For example, a single dose comprises about 5×10⁸CD19 CART cells. In embodiments, a lymphodepleting chemotherapy isadministered to the subject prior to, concurrently with, or afteradministration (e.g., infusion) of a CAR-expressing cell describedherein, e.g., a non-CD19 CAR-expressing cell. In embodiments, a CD19CART is administered to the subject prior to, concurrently with, orafter administration (e.g., infusion) of a non-CD19 CAR-expressing cell,e.g., a non-CD19 CAR-expressing cell described herein.

In some embodiments, a CAR-expressing cell described herein isadministered to a subject in combination with a interleukin-15 (IL-15)polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or acombination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g.,hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimericnon-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described in,e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299,U.S. 2012/0141413, and U.S. 2011/0081311, incorporated herein byreference. In embodiments, het-IL-15 is administered subcutaneously. Inembodiments, the subject has a cancer, e.g., solid cancer, e.g.,melanoma or colon cancer. In embodiments, the subject has a metastaticcancer.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like. CRS mayinclude clinical constitutional signs and symptoms such as fever,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.CRS may include clinical skin signs and symptoms such as rash. CRS mayinclude clinical gastrointestinal signs and symptoms such as nausea,vomiting and diarrhea. CRS may include clinical respiratory signs andsymptoms such as tachypnea and hypoxemia. CRS may include clinicalcardiovascular signs and symptoms such as tachycardia, widened pulsepressure, hypotension, increased cardiac output (early) and potentiallydiminished cardiac output (late). CRS may include clinical coagulationsigns and symptoms such as elevated d-dimer, hypofibrinogenemia with orwithout bleeding. CRS may include clinical renal signs and symptoms suchas azotemia. CRS may include clinical hepatic signs and symptoms such astransaminitis and hyperbilirubinemia. CRS may include clinicalneurologic signs and symptoms such as headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, and seizures.

Accordingly, the methods described herein can comprise administering aCAR-expressing cell described herein to a subject and furtheradministering one or more agents to manage elevated levels of a solublefactor resulting from treatment with a CAR-expressing cell. In oneembodiment, the soluble factor elevated in the subject is one or more ofIFN-γ, TNFα, IL-2 and IL-6. In an embodiment, the factor elevated in thesubject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 andfraktalkine. Therefore, an agent administered to treat this side effectcan be an agent that neutralizes one or more of these soluble factors.In one embodiment, the agent that neutralizes one or more of thesesoluble forms is an antibody or antigen binding fragment thereof.Examples of such agents include, but are not limited to a steroid (e.g.,corticosteroid), an inhibitor of TNFα, and an inhibitor of IL-6. Anexample of a TNFα inhibitor is an anti-TNFα antibody molecule such as,infliximab, adalimumab, certolizumab pegol, and golimumab. Anotherexample of a TNFα inhibitor is a fusion protein such as entanercept.Small molecule inhibitors of TNFα include, but are not limited to,xanthine derivatives (e.g. pentoxifylline) and bupropion. An example ofan IL-6 inhibitor is an anti-IL-6 antibody molecule or an anti-IL-6receptor antibody molecule such as tocilizumab (toc), sarilumab,elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038,VX30, ARGX-109, FE301, and FM101. In one embodiment, the anti-IL-6receptor antibody molecule is tocilizumab. An example of an IL-1R basedinhibitor is anakinra.

In one embodiment, the subject can be administered an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Inhibitory molecules, e.g., Programmed Death 1 (PD-1), can, insome embodiments, decrease the ability of a CAR-expressing cell to mountan immune effector response. Examples of inhibitory molecules includePD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA,RNA or protein level, can optimize a CAR-expressing cell performance. Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used to inhibitexpression of an inhibitory molecule in the CAR-expressing cell. In anembodiment the inhibitor is an shRNA. In an embodiment, the inhibitorymolecule is inhibited within a CAR-expressing cell. In theseembodiments, a dsRNA molecule that inhibits expression of the inhibitorymolecule is linked to the nucleic acid that encodes a component, e.g.,all of the components, of the CAR. In one embodiment, the inhibitor ofan inhibitory signal can be, e.g., an antibody or antibody fragment thatbinds to an inhibitory molecule. For example, the agent can be anantibody or antibody fragment that binds to PD-1, PD-L1, PD-L2 or CTLA4(e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketedas Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibodyavailable from Pfizer, formerly known as ticilimumab, CP-675,206).). Inan embodiment, the agent is an antibody or antibody fragment that bindsto TIM3. In an embodiment, the agent is an antibody or antibody fragmentthat binds to CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5). In anembodiment, the agent is an antibody or antibody fragment that binds toLAGS.

PD-1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol8:765-75). Two ligands for PD-1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD-1 (Freeman et a. 2000J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carteret al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094)Immune suppression can be reversed by inhibiting the local interactionof PD-1 with PD-L1. Antibodies, antibody fragments, and other inhibitorsof PD-1, PD-L1 and PD-L2 are available in the art and may be usedcombination with a cars of the present invention described herein. Forexample, nivolumab (also referred to as BMS-936558 or MDX1106;Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody whichspecifically blocks PD-1. Nivolumab (clone 5C4) and other humanmonoclonal antibodies that specifically bind to PD-1 are disclosed inU.S. Pat. No. 8,008,449 and WO2006/121168. Pidilizumab (CT-011; CureTech) is a humanized IgG1k monoclonal antibody that binds to PD-1.Pidilizumab and other humanized anti-PD-1 monoclonal antibodies aredisclosed in WO2009/101611. Pembrolizumab (formerly known aslambrolizumab, and also referred to as MK03475; Merck) is a humanizedIgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibodythat binds to PDL1, and inhibits interaction of the ligand with PD1.MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonalantibody that binds to PD-L1. MDPL3280A and other human monoclonalantibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.SPublication No.: 20120039906. Other anti-PD-L1 binding agents includeYW243.55.570 (heavy and light chain variable regions are shown in SEQ IDNOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to asBMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD-1 and B7-H1. Other anti-PD-1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649.

TIM-3 (T cell immunoglobulin-3) also negatively regulates T cellfunction, particularly in IFN-g-secreting CD4+ T helper 1 and CD8+ Tcytotoxic 1 cells, and plays a critical role in T cell exhaustion.Inhibition of the interaction between TIM3 and its ligands, e.g.,galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can increaseimmune response. Antibodies, antibody fragments, and other inhibitors ofTIM3 and its ligands are available in the art and may be usedcombination with a CD19 CAR described herein. For example, antibodies,antibody fragments, small molecules, or peptide inhibitors that targetTIM3 binds to the IgV domain of TIM3 to inhibit interaction with itsligands. Antibodies and peptides that inhibit TIM3 are disclosed inWO2013/006490 and US20100247521. Other anti-TIM3 antibodies includehumanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, CancerRes, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002,Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1are disclosed in US20130156774.

In other embodiments, the agent that enhances the activity of aCAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3,and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAMis an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodiesare described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or arecombinant form thereof, as described in, e.g., US 2004/0047858, U.S.Pat. No. 7,132,255 and WO 99/052552. In other embodiments, theanti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng etal. PLoS One. 2010 Sep. 2; 5(9). pii: e12529(DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 andCEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, carcinoembryonic antigen celladhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believedto mediate, at least in part, inhibition of an anti-tumor immuneresponse (see e.g., Markel et al. J Immunol. 2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71;Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al.Cancer Immunol Immunother. 2010 February; 59(2):215-30; Ortenberg et al.Mol Cancer Ther. 2012 June; 11(6):1300-10; Stern et al. J Immunol. 2005Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii:e12529). For example, CEACAM-1 has been described as a heterophilicligand for TIM-3 and as playing a role in TIM-3-mediated T celltolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014)Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1and TIM-3 has been shown to enhance an anti-tumor immune response inxenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, etal. (2014), supra). In other embodiments, co-blockade of CEACAM-1 andPD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251.Thus, CEACAM inhibitors can be used with the other immunomodulatorsdescribed herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) toenhance an immune response against a cancer, e.g., a melanoma, a lungcancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovariancancer, and other cancers as described herein.

LAG-3 (lymphocyte activation gene-3 or CD223) is a cell surface moleculeexpressed on activated T cells and B cells that has been shown to play arole in CD8+ T cell exhaustion. Antibodies, antibody fragments, andother inhibitors of LAG-3 and its ligands are available in the art andmay be used combination with a CD19 CAR described herein. For example,BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targetsLAG3. IMP701 (Immutep) is an antagonist LAG-3 antibody and IMP731(Immutep and GlaxoSmithKline) is a depleting LAG-3 antibody. Other LAG-3inhibitors include IMP321 (Immutep), which is a recombinant fusionprotein of a soluble portion of LAG3 and Ig that binds to MHC class IImolecules and activates antigen presenting cells (APC). Other antibodiesare disclosed, e.g., in WO2010/019570.

In some embodiments, the agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptidecomprising an intracellular signaling domain as described herein. Insome embodiments, the polypeptide that is associated with a positivesignal can include a costimulatory domain of CD28, CD27, ICOS, e.g., anintracellular signaling domain of CD28, CD27 and/or ICOS, and/or aprimary signaling domain, e.g., of CD3 zeta, e.g., described herein. Inone embodiment, the fusion protein is expressed by the same cell thatexpressed the CAR. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T cell that does not express a CAR of thepresent invention.

In one embodiment, the agent which enhances activity of a CAR-expressingcell described herein is miR-17-92.

In one embodiment, the agent which enhances activity of a CAR-describedherein is a cytokine. Cytokines have important functions related to Tcell expansion, differentiation, survival, and homeostasis. Cytokinesthat can be administered to the subject receiving a CAR-expressing celldescribed herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, andIL-21, or a combination thereof. In preferred embodiments, the cytokineadministered is IL-7, IL-15, or IL-21, or a combination thereof. Thecytokine can be administered once a day or more than once a day, e.g.,twice a day, three times a day, or four times a day. The cytokine can beadministered for more than one day, e.g. the cytokine is administeredfor 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or4 weeks. For example, the cytokine is administered once a day for 7days.

In embodiments, the cytokine is administered in combination withCAR-expressing T cells. The cytokine can be administered simultaneouslyor concurrently with the CAR-expressing T cells, e.g., administered onthe same day. The cytokine may be prepared in the same pharmaceuticalcomposition as the CAR-expressing T cells, or may be prepared in aseparate pharmaceutical composition. Alternatively, the cytokine can beadministered shortly after administration of the CAR-expressing T cells,e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days afteradministration of the CAR-expressing T cells. In embodiments where thecytokine is administered in a dosing regimen that occurs over more thanone day, the first day of the cytokine dosing regimen can be on the sameday as administration with the CAR-expressing T cells, or the first dayof the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5days, 6 days, or 7 days after administration of the CAR-expressing Tcells. In one embodiment, on the first day, the CAR-expressing T cellsare administered to the subject, and on the second day, a cytokine isadministered once a day for the next 7 days. In a preferred embodiment,the cytokine to be administered in combination with CAR-expressing Tcells is IL-7, IL-15, or IL-21.

In other embodiments, the cytokine is administered a period of timeafter administration of CAR-expressing cells, e.g., at least 2 weeks, 3weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or1 year or more after administration of CAR-expressing cells. In oneembodiment, the cytokine is administered after assessment of thesubject's response to the CAR-expressing cells. For example, the subjectis administered CAR-expressing cells according to the dosage andregimens described herein. The response of the subject to CAR-expressingcell therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks,10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, or 1 year or more after administration ofCAR-expressing cells, using any of the methods described herein,including inhibition of tumor growth, reduction of circulating tumorcells, or tumor regression. Subjects that do not exhibit a sufficientresponse to CAR-expressing cell therapy can be administered a cytokine.Administration of the cytokine to the subject that has sub-optimalresponse to the CAR-expressing cell therapy improves CAR-expressing cellefficacy or anti-cancer activity. In a preferred embodiment, thecytokine administered after administration of CAR-expressing cells isIL-7.

Combination with a Low Dose of an mTOR Inhibitor

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15, but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, or at least 70 but no more than 90%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 80%, at least10 but no more than 80%, at least 15, but no more than 80%, at least 20but no more than 80%, at least 30 but no more than 80%, at least 40 butno more than 80%, at least 50 but no more than 80%, or at least 60 butno more than 80%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 70%, at least10 but no more than 70%, at least 15, but no more than 70%, at least 20but no more than 70%, at least 30 but no more than 70%, at least 40 butno more than 70%, or at least 50 but no more than 70%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 60%, at least10 but no more than 60%, at least 15, but no more than 60%, at least 20but no more than 60%, at least 30 but no more than 60%, or at least 40but no more than 60%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 50%, at least10 but no more than 50%, at least 15, but no more than 50%, at least 20but no more than 50%, at least 30 but no more than 50%, or at least 40but no more than 50%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 40%, at least10 but no more than 40%, at least 15, but no more than 40%, at least 20but no more than 40%, at least 30 but no more than 40%, or at least 35but no more than 40%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 30%, at least10 but no more than 30%, at least 15, but no more than 30%, at least 20but no more than 30%, or at least 25 but no more than 30%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at least 1, 2, 3, 4or 5, but no more than 35, at least 1, 2, 3, 4 or 5 but no more than40%, or at least 1, 2, 3, 4 or 5 but no more than 45%.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than90%.

As is discussed herein, the extent of mTOR inhibition can be expressedas the extent of P70 S6 kinase inhibition, e.g., the extent of mTORinhibition can be determined by the level of decrease in P70 S6 kinaseactivity, e.g., by the decrease in phosphorylation of a P70 S6 kinasesubstrate. The level of mTOR inhibition can be evaluated by a methoddescribed herein, e.g. by the Boulay assay, or measurement ofphosphorylated S6 levels by western blot.

Exemplary mTOR Inhibitors

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment an mTOR inhibitor is anallosteric inhibitor. In an embodiment an mTOR inhibitor is a catalyticinhibitor.

Allosteric mTOR inhibitors include the neutral tricyclic compoundrapamycin (sirolimus), rapamycin-related compounds, that is compoundshaving structural and functional similarity to rapamycin including,e.g., rapamycin derivatives, rapamycin analogs (also referred to asrapalogs) and other macrolide compounds that inhibit mTOR activity.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,O-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as, everolimus as described in U.S. Pat. No. 5,665,772 andWO94/09010 the contents of which are incorporated by reference. Othersuitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4′S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(25)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin and40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in U.S. RE44,768, e.g.temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, preferably (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807 the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described inUS2005/0101624 the contents of which are incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone

Further examples of allosteric mTOR inhibitors include sirolimus(rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTor inhibitors include zotarolimus(ABT578) and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more effective inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile,or the monotosylate salt form. the synthesis of BEZ235 is described inWO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C. M., etal., Cancer Res, 2010, 70(1), 288-298) which has the chemical name{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol;3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide(WO09104019);3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine(WO10051043 and WO2013023184); AN-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide(WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem.,2010, 53, 2636-2645) which has the chemical name1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea;GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has thechemical name2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide;5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(WO10114484);(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazoquinolin-2(3H)-ylidene)cyanamide (WO12007926).

Further examples of catalytic mTOR inhibitors include8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J.,2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammaliantarget of rapamycin (mTOR).) WYE-354 is another example of a catalyticmTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivoActivity of Novel ATP-Competitive and Selective Inhibitors of theMammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240).

mTOR inhibitors useful according to the present invention also includeprodrugs, derivatives, pharmaceutically acceptable salts, or analogsthereof of any of the foregoing.

mTOR inhibitors, such as RAD001, may be formulated for delivery based onwell-established methods in the art based on the particular dosagesdescribed herein. In particular, U.S. Pat. No. 6,004,973 (incorporatedherein by reference) provides examples of formulations useable with themTOR inhibitors described herein.

Evaluation of mTor Inhibition

mTOR phosphorylates the kinase P70 S6, thereby activating P70 S6 kinaseand allowing it to phosphorylate its substrate. The extent of mTORinhibition can be expressed as the extent of P70 S6 kinase inhibition,e.g., the extent of mTOR inhibition can be determined by the level ofdecrease in P70 S6 kinase activity, e.g., by the decrease inphosphorylation of a P70 S6 kinase substrate. One can determine thelevel of mTOR inhibition, by measuring P70 S6 kinase activity (theability of P70 S6 kinase to phosphorylate a substrate), in the absenceof inhibitor, e.g., prior to administration of inhibitor, and in thepresences of inhibitor, or after the administration of inhibitor. Thelevel of inhibition of P70 S6 kinase gives the level of mTOR inhibition.Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measuredby P70 S6 kinase activity, is inhibited by 40%. The extent or level ofinhibition referred to herein is the average level of inhibition overthe dosage interval. By way of example, if the inhibitor is given onceper week, the level of inhibition is given by the average level ofinhibition over that interval, namely a week.

Boulay et al., Cancer Res, 2004, 64:252-61, hereby incorporated byreference, teaches an assay that can be used to assess the level of mTORinhibition (referred to herein as the Boulay assay). In an embodiment,the assay relies on the measurement of P70 S6 kinase activity frombiological samples before and after administration of an mTOR inhibitor,e.g., RAD001. Samples can be taken at preselected times after treatmentwith an mTOR inhibitor, e.g., 24, 48, and 72 hours after treatment.Biological samples, e.g., from skin or peripheral blood mononuclearcells (PBMCs) can be used. Total protein extracts are prepared from thesamples. P70 S6 kinase is isolated from the protein extracts byimmunoprecipitation using an antibody that specifically recognizes theP70 S6 kinase. Activity of the isolated P70 S6 kinase can be measured inan in vitro kinase assay. The isolated kinase can be incubated with 40Sribosomal subunit substrates (which is an endogenous substrate of P70 S6kinase) and gamma-³²P under conditions that allow phosphorylation of thesubstrate. Then the reaction mixture can be resolved on an SDS-PAGE gel,and ³²P signal analyzed using a PhosphorImager. A ³²P signalcorresponding to the size of the 40S ribosomal subunit indicatesphosphorylated substrate and the activity of P70 S6 kinase. Increasesand decreases in kinase activity can be calculated by quantifying thearea and intensity of the ³²P signal of the phosphorylated substrate(e.g., using ImageQuant, Molecular Dynamics), assigning arbitrary unitvalues to the quantified signal, and comparing the values from afteradministration with values from before administration or with areference value. For example, percent inhibition of kinase activity canbe calculated with the following formula: 1−(value obtained afteradministration/value obtained before administration)×100. As describedabove, the extent or level of inhibition referred to herein is theaverage level of inhibition over the dosage interval.

Methods for the evaluation of kinase activity, e.g., P70 S6 kinaseactivity, are also provided in U.S. Pat. No. 7,727,950, herebyincorporated by reference.

The level of mTOR inhibition can also be evaluated by a change in theration of PD1 negative to PD1 positive T cells. T cells from peripheralblood can be identified as PD1 negative or positive by art-knownmethods.

Low-Dose mTOR Inhibitors

Methods described herein use low, immune enhancing, dose mTORinhibitors, doses of mTOR inhibitors, e.g., allosteric mTOR inhibitors,including rapalogs such as RAD001. In contrast, levels of inhibitor thatfully or near fully inhibit the mTOR pathway are immunosuppressive andare used, e.g., to prevent organ transplant rejection. In addition, highdoses of rapalogs that fully inhibit mTOR also inhibit tumor cell growthand are used to treat a variety of cancers (See, e.g., Antineoplasticeffects of mammalian target of rapamycine inhibitors. Salvadori M. WorldJ Transplant. 2012 Oct. 24; 2(5):74-83; Current and Future TreatmentStrategies for Patients with Advanced Hepatocellular Carcinoma: Role ofmTOR Inhibition. Finn R S. Liver Cancer. 2012 November; 1(3-4):247-256;Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A,Cornellà H, Villanueva A. Liver Cancer. 2012 September; 1(2):83-93;Targeted cancer therapy—Are the days of systemic chemotherapy numbered?Joo W D, Visintin I, Mor G. Maturitas. 2013 Sep. 20; Role of natural andadaptive immunity in renal cell carcinoma response to VEGFR-TKIs andmTOR inhibitor. Santoni M, Berardi R, Amantini C, Burattini L, SantiniD, Santoni G, Cascinu S. Int J Cancer. 2013 Oct. 2).

The present invention is based, at least in part, on the surprisingfinding that doses of mTOR inhibitors well below those used in currentclinical settings had a superior effect in increasing an immune responsein a subject and increasing the ratio of PD-1 negative T cells/PD-1positive T cells. It was surprising that low doses of mTOR inhibitors,producing only partial inhibition of mTOR activity, were able toeffectively improve immune responses in human subjects and increase theratio of PD-1 negative T cells/PD-1 positive T cells.

Alternatively, or in addition, without wishing to be bound by anytheory, it is believed that low, a low, immune enhancing, dose of anmTOR inhibitor can increase naive T cell numbers, e.g., at leasttransiently, e.g., as compared to a non-treated subject. Alternativelyor additionally, again while not wishing to be bound by theory, it isbelieved that treatment with an mTOR inhibitor after a sufficient amountof time or sufficient dosing results in one or more of the following:

an increase in the expression of one or more of the following markers:CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on memory T cells,e.g., memory T cell precursors;

a decrease in the expression of KLRG1, e.g., on memory T cells, e.g.,memory T cell precursors; and

an increase in the number of memory T cell precursors, e.g., cells withany one or combination of the following characteristics: increasedCD62L^(high) increased CD127^(high), increased CD27⁺, decreased KLRG1,and increased BCL2;

and wherein any of the changes described above occurs, e.g., at leasttransiently, e.g., as compared to a non-treated subject (Araki, K et al.(2009) Nature 460:108-112). Memory T cell precursors are memory T cellsthat are early in the differentiation program. For example, memory Tcells have one or more of the following characteristics: increasedCD62L^(high), increased CD127^(high) increased CD27⁺, decreased KLRG1,and/or increased BCL2.

In an embodiment, the invention relates to a composition, or dosageform, of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., arapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, which,when administered on a selected dosing regimen, e.g., once daily or onceweekly, is associated with: a level of mTOR inhibition that is notassociated with complete, or significant immune suppression, but isassociated with enhancement of the immune response.

An mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., a rapalog,rapamycin, or RAD001, or a catalytic mTOR inhibitor, can be provided ina sustained release formulation. Any of the compositions or unit dosageforms described herein can be provided in a sustained releaseformulation. In some embodiments, a sustained release formulation willhave lower bioavailability than an immediate release formulation. E.g.,in embodiments, to attain a similar therapeutic effect of an immediaterelease formulation a sustained release formulation will have from about2 to about 5, about 2.5 to about 3.5, or about 3 times the amount ofinhibitor provided in the immediate release formulation.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per week, having 0.1 to 20, 0.5 to 10, 2.5to 7.5, 3 to 6, or about 5, mgs per unit dosage form, are provided. Foronce per week administrations, these immediate release formulationscorrespond to sustained release forms, having, respectively, 0.3 to 60,1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of an mTOR inhibitor,e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. Inembodiments both forms are administered on a once/week basis.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per day, having 0.005 to 1.5, 0.01 to 1.5,0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5, 0.6 to 1.5,0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or about 0.5 mgs perunit dosage form, are provided. For once per day administrations, theseimmediate release forms correspond to sustained release forms, having,respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to4.5, 1.2 to 4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to4.5, 0.9 to 1.8, or about 1.5 mgs of an mTOR inhibitor, e.g., anallosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per weekadministrations, these immediate release forms correspond to sustainedrelease forms, having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30,6 to 12, or about 10 mgs of an mTOR inhibitor, e.g., an allosteric mTORinhibitor, e.g., rapamycin or RAD001.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per day, having 0.01 to 1.0 mgs per unitdosage form, are provided. For once per day administrations, theseimmediate release forms correspond to sustained release forms, having,respectively, 0.03 to 3 mgs of an mTOR inhibitor, e.g., an allostericmTOR inhibitor, e.g., rapamycin or RAD001. For once per weekadministrations, these immediate release forms correspond to sustainedrelease forms, having, respectively, 0.2 to 20 mgs of an mTOR inhibitor,e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001.

In an embodiment, immediate release forms, e.g., of RAD001, typicallyused for one administration per week, having 0.5 to 5.0 mgs per unitdosage form, are provided. For once per week administrations, theseimmediate release forms correspond to sustained release forms, having,respectively, 1.5 to 15 mgs of an mTOR inhibitor, e.g., an allostericmTOR inhibitor, e.g., rapamycin or RAD001.

As described above, one target of the mTOR pathway is the P70 S6 kinase.Thus, doses of mTOR inhibitors which are useful in the methods andcompositions described herein are those which are sufficient to achieveno greater than 80% inhibition of P70 S6 kinase activity relative to theactivity of the P70 S6 kinase in the absence of an mTOR inhibitor, e.g.,as measured by an assay described herein, e.g., the Boulay assay. In afurther aspect, the invention provides an amount of an mTOR inhibitorsufficient to achieve no greater than 38% inhibition of P70 S6 kinaseactivity relative to P70 S6 kinase activity in the absence of an mTORinhibitor.

In one aspect the dose of mTOR inhibitor useful in the methods andcompositions of the invention is sufficient to achieve, e.g., whenadministered to a human subject, 90+/−5% (i.e., 85-95%), 89+/−5%,88+/−5%, 87+/−5%, 86+/−5%, 85+/−5%, 84+/−5%, 83+/−5%, 82+/−5%, 81+/−5%,80+/−5%, 79+/−5%, 78+/−5%, 77+/−5%, 76+/−5%, 75+/−5%, 74+/−5%, 73+/−5%,72+/−5%, 71+/−5%, 70+/−5%, 69+/−5%, 68+/−5%, 67+/−5%, 66+/−5%, 65+/−5%,64+/−5%, 63+/−5%, 62+/−5%, 61+/−5%, 60+/−5%, 59+/−5%, 58+/−5%, 57+/−5%,56+/−5%, 55+/−5%, 54+/−5%, 54+/−5%, 53+/−5%, 52+/−5%, 51+/−5%, 50+/−5%,49+/−5%, 48+/−5%, 47+/−5%, 46+/−5%, 45+/−5%, 44+/−5%, 43+/−5%, 42+/−5%,41+/−5%, 40+/−5%, 39+/−5%, 38+/−5%, 37+/−5%, 36+/−5%, 35+/−5%, 34+/−5%,33+/−5%, 32+/−5%, 31+/−5%, 30+/−5%, 29+/−5%, 28+/−5%, 27+/−5%, 26+/−5%,25+/−5%, 24+/−5%, 23+/−5%, 22+/−5%, 21+/−5%, 20+/−5%, 19+/−5%, 18+/−5%,17+/−5%, 16+/−5%, 15+/−5%, 14+/−5%, 13+/−5%, 12+/−5%, 11+/−5%, or10+/−5%, inhibition of P70 S6 kinase activity, e.g., as measured by anassay described herein, e.g., the Boulay assay.

P70 S6 kinase activity in a subject may be measured using methods knownin the art, such as, for example, according to the methods described inU.S. Pat. No. 7,727,950, by immunoblot analysis of phosphoP70 S6K levelsand/or phosphoP70 S6 levels or by in vitro kinase activity assays.

As used herein, the term “about” in reference to a dose of mTORinhibitor refers to up to a +/−10% variability in the amount of mTORinhibitor, but can include no variability around the stated dose.

In some embodiments, the invention provides methods comprisingadministering to a subject an mTOR inhibitor, e.g., an allostericinhibitor, e.g., RAD001, at a dosage within a target trough level. Insome embodiments, the trough level is significantly lower than troughlevels associated with dosing regimens used in organ transplant andcancer patients. In an embodiment mTOR inhibitor, e.g., RAD001, orrapamycin, is administered to result in a trough level that is less than½, ¼, 1/10, or 1/20 of the trough level that results inimmunosuppression or an anticancer effect. In an embodiment mTORinhibitor, e.g., RAD001, or rapamycin, is administered to result in atrough level that is less than ½, ¼, 1/10, or 1/20 of the trough levelprovided on the FDA approved packaging insert for use inimmunosuppression or an anticancer indications.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.1 to 10 ng/ml, 0.1to 5 ng/ml, 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.2 to 10 ng/ml, 0.2to 5 ng/ml, 0.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g. an, allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.3 to 10 ng/ml, 0.3to 5 ng/ml, 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.4 to 10 ng/ml, 0.4to 5 ng/ml, 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 0.5 to 10 ng/ml, 0.5to 5 ng/ml, 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml.

In an embodiment a method disclosed herein comprises administering to asubject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001,at a dosage that provides a target trough level of 1 to 10 ng/ml, 1 to 5ng/ml, 1 to 3 ng/ml, or 1 to 2 ng/ml.

As used herein, the term “trough level” refers to the concentration of adrug in plasma just before the next dose, or the minimum drugconcentration between two doses.

In some embodiments, a target trough level of RAD001 is in a range ofbetween about 0.1 and 4.9 ng/ml. In an embodiment, the target troughlevel is below 3 ng/ml, e.g., is between 0.3 or less and 3 ng/ml. In anembodiment, the target trough level is below 3 ng/ml, e.g., is between0.3 or less and 1 ng/ml.

In a further aspect, the invention can utilize an mTOR inhibitor otherthan RAD001 in an amount that is associated with a target trough levelthat is bioequivalent to the specified target trough level for RAD001.In an embodiment, the target trough level for an mTOR inhibitor otherthan RAD001, is a level that gives the same level of mTOR inhibition(e.g., as measured by a method described herein, e.g., the inhibition ofP70 S6) as does a trough level of RAD001 described herein.

Pharmaceutical Compositions: mTOR Inhibitors

In one aspect, the present invention relates to pharmaceuticalcompositions comprising an mTOR inhibitor, e.g., an mTOR inhibitor asdescribed herein, formulated for use in combination with CAR cellsdescribed herein.

In some embodiments, the mTOR inhibitor is formulated for administrationin combination with an additional, e.g., as described herein.

In general, compounds of the invention will be administered intherapeutically effective amounts as described above via any of theusual and acceptable modes known in the art, either singly or incombination with one or more therapeutic agents.

The pharmaceutical formulations may be prepared using conventionaldissolution and mixing procedures. For example, the bulk drug substance(e.g., an mTOR inhibitor or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described herein. The mTOR inhibitor is typicallyformulated into pharmaceutical dosage forms to provide an easilycontrollable dosage of the drug and to give the patient an elegant andeasily handleable product.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Where an mTOR inhibitor is administered in combinationwith (either simultaneously with or separately from) another agent asdescribed herein, in one aspect, both components can be administered bythe same route (e.g., parenterally). Alternatively, another agent may beadministered by a different route relative to the mTOR inhibitor. Forexample, an mTOR inhibitor may be administered orally and the otheragent may be administered parenterally.

Sustained Release

mTOR inhibitors, e.g., allosteric mTOR inhibitors or catalytic mTORinhibitors, disclosed herein can be provided as pharmaceuticalformulations in form of oral solid dosage forms comprising an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, which satisfyproduct stability requirements and/or have favorable pharmacokineticproperties over the immediate release (IR) tablets, such as reducedaverage plasma peak concentrations, reduced inter- and intra-patientvariability in the extent of drug absorption and in the plasma peakconcentration, reduced C_(max)/C_(min) ratio and/or reduced foodeffects. Provided pharmaceutical formulations may allow for more precisedose adjustment and/or reduce frequency of adverse events thus providingsafer treatments for patients with an mTOR inhibitor disclosed herein,e.g., rapamycin or RAD001.

In some embodiments, the present disclosure provides stable extendedrelease formulations of an mTOR inhibitor disclosed herein, e.g.,rapamycin or RAD001, which are multi-particulate systems and may havefunctional layers and coatings.

The term “extended release, multi-particulate formulation as used hereinrefers to a formulation which enables release of an mTOR inhibitordisclosed herein, e.g., rapamycin or RAD001, over an extended period oftime e.g. over at least 1, 2, 3, 4, 5 or 6 hours. The extended releaseformulation may contain matrices and coatings made of specialexcipients, e.g., as described herein, which are formulated in a manneras to make the active ingredient available over an extended period oftime following ingestion.

The term “extended release” can be interchangeably used with the terms“sustained release” (SR) or “prolonged release”. The term “extendedrelease” relates to a pharmaceutical formulation that does not releaseactive drug substance immediately after oral dosing but over an extendedin accordance with the definition in the pharmacopoeias Ph. Eur. (7^(th)edition) mongraph for tablets and capsules and USP general chapter<1151> for pharmaceutical dosage forms. The term “Immediate Release”(IR) as used herein refers to a pharmaceutical formulation whichreleases 85% of the active drug substance within less than 60 minutes inaccordance with the definition of “Guidance for Industry: “DissolutionTesting of Immediate Release Solid Oral Dosage Forms” (FDA CDER, 1997).In some embodiments, the term “immediate release” means release ofeverolismus from tablets within the time of 30 minutes, e.g., asmeasured in the dissolution assay described herein.

Stable extended release formulations of an mTOR inhibitor disclosedherein, e.g., rapamycin or RAD001, can be characterized by an in-vitrorelease profile using assays known in the art, such as a dissolutionassay as described herein: a dissolution vessel filled with 900 mLphosphate buffer pH 6.8 containing sodium dodecyl sulfate 0.2% at 37° C.and the dissolution is performed using a paddle method at 75 rpmaccording to USP by according to USP testing monograph 711, and Ph.Eur.testing monograph 2.9.3. respectively.

In some embodiments, stable extended release formulations of an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, release the mTORinhibitor in the in-vitro release assay according to following releasespecifications:

0.5 h: <45%, or <40, e.g., <30%

1 h: 20-80%, e.g., 30-60%

2 h: >50%, or >70%, e.g., >75%

3 h: >60%, or >65%, e.g., >85%, e.g., >90%.

In some embodiments, stable extended release formulations of an mTORinhibitor disclosed herein, e.g., rapamycin or RAD001, release 50% ofthe mTOR inhibitor not earlier than 45, 60, 75, 90, 105 min or 120 minin the in-vitro dissolution assay.

Biopolymer Delivery Methods

In some embodiments, one or more CAR-expressing cells as disclosedherein can be administered or delivered to the subject via a biopolymerscaffold, e.g., a biopolymer implant. Biopolymer scaffolds can supportor enhance the delivery, expansion, and/or dispersion of theCAR-expressing cells described herein. A biopolymer scaffold comprises abiocompatible (e.g., does not substantially induce an inflammatory orimmune response) and/or a biodegradable polymer that can be naturallyoccurring or synthetic.

Examples of suitable biopolymers include, but are not limited to, agar,agarose, alginate, alginate/calcium phosphate cement (CPC),beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose),cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acidcollagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate)(PHBHHx), poly(lactide), poly(caprolactone) (PCL),poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO),poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO),polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate,alone or in combination with any other polymer composition, in anyconcentration and in any ratio. The biopolymer can be augmented ormodified with adhesion- or migration-promoting molecules, e.g.,collagen-mimetic peptides that bind to the collagen receptor oflymphocytes, and/or stimulatory molecules to enhance the delivery,expansion, or function, e.g., anti-cancer activity, of the cells to bedelivered. The biopolymer scaffold can be an injectable, e.g., a gel ora semi-solid, or a solid composition.

In some embodiments, CAR-expressing cells described herein are seededonto the biopolymer scaffold prior to delivery to the subject. Inembodiments, the biopolymer scaffold further comprises one or moreadditional therapeutic agents described herein (e.g., anotherCAR-expressing cell, an antibody, or a small molecule) or agents thatenhance the activity of a CAR-expressing cell, e.g., incorporated orconjugated to the biopolymers of the scaffold. In embodiments, thebiopolymer scaffold is injected, e.g., intratumorally, or surgicallyimplanted at the tumor or within a proximity of the tumor sufficient tomediate an anti-tumor effect. Additional examples of biopolymercompositions and methods for their delivery are described in Stephan etal., Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions of the present invention may comprise aCAR-expressing cell, e.g., a plurality of CAR-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the immune effector cells (e.g., T cells, NK cells) describedherein may be administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, in some instances 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. T cell compositions may also beadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

In certain aspects, it may be desired to administer activated immuneeffector cells (e.g., T cells, NK cells) to a subject and thensubsequently redraw blood (or have an apheresis performed), activateimmune effector cells (e.g., T cells, NK cells) therefrom according tothe present invention, and reinfuse the patient with these activated andexpanded immune effector cells (e.g., T cells, NK cells). This processcan be carried out multiple times every few weeks. In certain aspects,immune effector cells (e.g., T cells, NK cells) can be activated fromblood draws of from 10 cc to 400 cc. In certain aspects, immune effectorcells (e.g., T cells, NK cells) are activated from blood draws of 20 cc,30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the T cell compositions of the presentinvention are administered by i.v. injection. The compositions of immuneeffector cells (e.g., T cells, NK cells) may be injected directly into atumor, lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukopheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates may be expanded by methods known in the art and treated suchthat one or more CAR constructs of the invention may be introduced,thereby creating a CAR T cell of the invention. Subjects in need thereofmay subsequently undergo standard treatment with high dose chemotherapyfollowed by peripheral blood stem cell transplantation. In certainaspects, following or concurrent with the transplant, subjects receivean infusion of the expanded CAR T cells of the present invention. In anadditional aspect, expanded cells are administered before or followingsurgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells(e.g., T cells, NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of CAR immuneeffector cells (e.g., T cells, NK cells) of the invention, and one ormore subsequent administrations of the CAR immune effector cells (e.g.,T cells, NK cells) of the invention, wherein the one or more subsequentadministrations are administered less than 15 days, e.g., 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of theCAR immune effector cells (e.g., T cells, NK cells) of the invention areadministered to the subject (e.g., human) per week, e.g., 2, 3, or 4administrations of the CAR immune effector cells (e.g., T cells, NKcells) of the invention are administered per week. In one embodiment,the subject (e.g., human subject) receives more than one administrationof the CAR immune effector cells (e.g., T cells, NK cells) per week(e.g., 2, 3 or 4 administrations per week) (also referred to herein as acycle), followed by a week of no CAR immune effector cells (e.g., Tcells, NK cells) administrations, and then one or more additionaladministration of the CAR immune effector cells (e.g., T cells, NKcells) (e.g., more than one administration of the CAR immune effectorcells (e.g., T cells, NK cells) per week) is administered to thesubject. In another embodiment, the subject (e.g., human subject)receives more than one cycle of CAR immune effector cells (e.g., Tcells, NK cells), and the time between each cycle is less than 10, 9, 8,7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells(e.g., T cells, NK cells) are administered every other day for 3administrations per week. In one embodiment, the CAR immune effectorcells (e.g., T cells, NK cells) of the invention are administered for atleast two, three, four, five, six, seven, eight or more weeks.

In one aspect, CAR-expressing cells of the present inventions aregenerated using lentiviral viral vectors, such as lentivirus. Cells,e.g., CARTs, generated that way will have stable CAR expression.

In one aspect, CAR-expressing cells, e.g., CARTs, are generated using aviral vector such as a gammaretroviral vector, e.g., a gammaretroviralvector described herein. CARTs generated using these vectors can havestable CAR expression.

In one aspect, CARTs transiently express CAR vectors for 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expressionof CARs can be effected by RNA CAR vector delivery. In one aspect, theCAR RNA is transduced into the T cell by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing CAR immune effector cells (e.g., T cells, NKcells) (particularly with murine scFv bearing CARTs) is anaphylaxisafter multiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CART infusion breaks should not last more than tento fourteen days.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: PD-1 CAR

In one embodiment, the extracellular domain (ECD) of inhibitorymolecules, e.g., Programmed Death 1 (PD-1), can be fused to atransmembrane domain and intracellular signaling domains such as 4-1BBand CD3 zeta. In one embodiment, the PD-1 CAR can be used alone. In oneembodiment, the PD-1 CAR can be used in combination with another CAR,e.g., CD19CAR. In one embodiment, the PD-1 CAR improves the persistenceof the T cell. In one embodiment, the CAR is a PD-1 CAR comprising theextracellular domain of PD-1 indicated as underlined in SEQ ID NO: 26(PD-1 domain is underlined)

SEQ ID NO: 26 Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeshaelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldlargrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr

The corresponding nucleotide sequence for the PD-1 CAR is shown below,with the PD-1 ECD underlined below in SEQ ID NO: 27 (PD-1 domain isunderlined)

SEQ ID NO: 27 Atggccctccctgtcactgccctgatctccccctcgcactcctgctccacgccgctagaccacccggatggtactggactctccggatcgcccgtggaatcccccaaccactcaccggcactcaggagtgactgagggcgataatgcgaccacacgtgctcgactccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtaccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatagggctcctctcgccggaacttgtggcgtgctccactgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagatctgtacattacaagcagccatcatgaggcccgtgcaaaccacccaggaggaggacggagctcctgccggaccccgaagaggaagaaggaggagcgagctgcgcgtgaagactcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcag gccatccccctcgc

Other examples of inhibitory molecules in include PD1, CTLA4, TIM3,LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. PD1 is aninhibitory member of the CD28 family of receptors that also includesCD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, Tcells and myeloid cells (Agata et al. 1996 Int Immunol 8:765-75). Twoligands for PD1, PD-L1 and PD-L2 have been shown to downregulate T cellactivation upon binding to PD1 (Freeman et a. 2000 J Exp Med192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al.2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Donget al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol.Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD1 with PD-L1.

Example 2: Decreasing the Affinity of CAR Increases Therapeutic Efficacy

Adoptive cell therapy (ACT) with CAR engineered 1′ cells can target andkill widespread malignant cells thereby inducing durable clinicalresponses in treating some hematopoietic malignancies (Kochenderfer, J.N., et al. (2010) Blood 116:4099-4102; Porter, D. L., et al. (2011) NEngl J Med 365:725-733; and Brentjens, R. J., et al. (2013) Sci TranslMed 5:177ra138). However, many commonly targeted tumor antigens are alsoexpressed by healthy tissues and on target off tumor toxicity from Tcell-mediated destruction of normal tissue has limited the developmentand adoption of this otherwise promising type of cancer therapy. Recentreports on severe adverse events associated with treatment of cancerpatients with CAR- or TCR-engineered T lymphocytes further illustratethe importance of target selection for safe and efficient therapy(Lamers et al., 2006, J Clin Oncol. 24:e20; Parkhurst et al., 2011,Molecular therapy: the journal of the American Society of Gene Therapy.19:620-6; Morgan et al., 2013, J Immunotherapy. 36:133-151; Linette etal., 2013, Blood. 122:863-71). In specific, the targeting of ErbB2(Her2/neu or CD340) with high affinity CARTs led to serious toxicity dueto target recognition on normal cardiopulmonary tissue (Morgan et al.,2013, Mol Therapy. 18:843-851), and similarly, the presence ofrelatively high levels of EGFR in healthy skin leads to dose-limitingskin toxicity (Perez-Soler et al., 2010, J Clin Oncol. 23:5235-46).

Selecting highly tissue-restricted antigens, cancer testis antigens,mutated gene products or viral proteins as targets could significantlyimprove the safety profile of using CART cells. However, none of theseantigens is present with high frequency in common cancers,constitutively expressed exclusively by malignant cells, functionallyimportant for tumor growth, and targetable with CART. Most of thetop-ranked target antigens that could be targeted by CART are expressedin potentially important normal tissues, such as ErbB, EGFR, MUC1, PSMA,and GD2 (Cheever et al., 2009, Clinical Cancer Research. 15:5323-37).Current strategies for generating CARs consist of selecting scFv withhigh affinity, as previous studies have shown that the activationthreshold inversely correlated with the affinity of the scFv(Chmielewski et al., 2004, J Clin Oncol. 173:7647-53; and Hudecek etal., 2013, Clinical Cancer Research. 19:3153-64. Studies indicate thatthe costimulatory domain of CARs does not influence the activationthreshold (Chmielewski et al., 2011, Gene Therapy. 18:62-72). After TCRstimulation there is a narrow window of affinity for optimal T cellactivation, and increasing the affinity of the TCRs does not necessarilyimprove treatment efficacy (Zhong et al., 2013, Proc Natl Acad Sci USA.110:6973-8; and Schmid et al., 2010, J Immunol. 184:4936-46).

In this example, it was determined whether equipping T cells with highaffinity scFv may limit the utility of CARs, due to poor discriminationof the CART for tumors and normal tissues that express the same antigenat lower levels. It was also determined whether fine-tuning the affinityof the scFv could increase the ability of CART cells to discriminatetumors from normal tissues expressing the same antigen at lower levels.CARs with affinities against two validated targets, ErbB2 and EGFR,which are amplified or overexpressed in variety of cancers but are alsoexpressed, at lower levels by normal tissues, were tested extensivelyagainst multiple tumor lines, as well as primary cell lines from normaltissues and organs. It was found that decreasing the affinity of thescFv could significantly increase the therapeutic index of CARs whilemaintaining robust antitumor efficacy.

The following materials and methods were used in the experimentsdescribed in this example:

Cell Lines and Primary Human Lymphocytes

SK-BR3, SK-OV3, BT-474, MCF7, MDA231, MDA468, HCC2281, MDA-361, MDA-453,HCC-1419, HCC-1569, UACC-812, LnCap, MDA-175, MCF-10A, HCC38 and HG261cell lines were purchased from American Type Culture Collection andcultured as instructed. Seven primary cell lines (keratinocytes,osteoblast, renal epithelial, pulmonary artery endothelial cells,pulmonary artery smooth muscle, neural progenitor, CD34+ enriched PBMC)were obtained from Promocell and cultured according to their protocols.Primary lymphocytes were isolated from normal donors by the Universityof Pennsylvania Human Immunology Core and cultured in R10 medium (RPMI1640 supplemented with 10% fetal calf serum; Invitrogen). Primarylymphocytes were stimulated with microbeads coated with CD3 and CD28stimulatory antibodies (Life Technologies, Grand Island, N.Y., Catalog)as described (Barrett et al., 2009, Proc Nat Acad Sci USA, 106:3360). Tcells were cryopreserved at day 10 in a solution of 90% fetal calf serumand 10% dimethylsulfoxide (DMSO) at 1×10⁸ cells/vial.

Generation of CAR Constructs for mRNA Electroporation and LentiviralTransduction.

CAR scFv domains against ErbB2 or EGFR were synthesized and/or amplifiedby PCR, based on sequencing information provided by the relevantpublications (Carter et al., 1992, Proc Nat Acad Sci USA, 89:4285; Zhouet al., 2007, J Mol Bio, 371:934), linked to CD8 transmembrane domainand 4-1BB and CD3Z intracellular signaling domains, and subcloned intopGEM.64A RNA based vector (Zhao et al., 2010, Cancer Res, 70:9053) orpTRPE lentiviral vectors (Carpenito et al., 2009, Proc Nat Acad Sci USA,106:3360.

Biacore Assay

Biotinylated ErbB2 was mobilized to a streptavidin coated sensor chip ata density of 200 RU. Binding affinity of the parental 4D5 antibody(Carter et al., 1992, Proc Nat Acad Sci USA, 89:4285) were compared torecombinant scFv. The purity and atomic mass of the scFv were verifiedby liquid chromatography-mass spectrometry. ScFv samples were serialdiluted 3-fold and injected over the chip at a constant flow rate.Association and dissociation rates of the protein complex were monitoredfor 270 s and 400 s, respectively. Double referencing was performedagainst a blank immobilized flow cell and a buffer blank and the datawas fit using a 1:1 Langmuir model or steady state affinity whereappropriate with the Biacore T200 evaluation software.

mRNA In Vitro Transcription and T Cell Electroporation

T7 mScript systems kit (CellScript) was used to generate IVT RNA.CD3/CD28 bead stimulated T cells were electroporated with IVT RNA usingBTX EM830 (Harvard Apparatus BTX) as previously described (Zhao et al.,2010, Cancer Res, 70:9053). Briefly, T cells were washed three times andresuspended in OPTI-MEM (Invitrogen) at a final concentration of 1-3×10⁸cells/ml. Subsequently, 0.1 ml of cells were mixed with 10 ug IVT RNA(or as indicated) and electroporated in a 2 mm cuvette.

Flow Cytometry Analysis

Antibodies were obtained from the following suppliers: anti-human CD3(BD Biosciences, 555335), anti-human CD8 (BD Biosciences 555366),anti-human CD107a (BD Biosciences 555801), anti-human CD137 (BDBiosciences 555956). Cell surface expression of ErbB2 was detected bybiotinylated anti-ErbB2 Affibody (Abcam, ab31890), and EGFR by FITCconjugated anti-EGFR affibody (Abcam, ab81872). ErbB2, EGFR and CD19specific CAR T cell expression were detected by ErbB2-Fc fusion protein(R&D system, 1129-ER), EGFR-Fc fusion protein and biotin-labeledpolyclonal goat anti-mouse F(ab)2 antibodies (Jackson Immunoresearch,115-066-072) respectively, incubated at 4° C. for 25 minutes and washedtwice (PBS with 2% FBS). Samples were then stained with PE-conjugatedanti-human IgG Fc Ab (eBioscience, 12-4998-82) or phycoerythrin-labeledstreptavidin (eBioscience, 17-4317-82), incubated at 4° C. for 25minutes and washed once. Flow cytometry acquisition was performed oneither a BD FacsCalibur or Accuri C6 Cytometer (BD Biosciences).Analysis was performed using FlowJo software (Treestar).

ELISA Assays

Target cells were washed and suspended at 1×10⁶ cells/ml in R10 medium(RPMI 1640 supplemented with 10% fetal calf serum; Invitrogen). 100 uleach target cell type were added in duplicate to a 96 well round bottomplate (Corning). Effector T cells were washed, and re-suspended at 1×10⁶cells/ml in R10 medium and then 100 ul of T cells were combined withtarget cells in the indicated wells. In addition, wells containing Tcells alone were prepared. The plates were incubated at 37° C. for 18 to20 hours. After the incubation, supernatant was harvested and subjectedto an ELISA assay (eBioscience, 88-7316-77; 88-7025-77).

CD107a Staining

Cells were plated at an E:T of 1:1 (1×10⁵ effectors: 1×10⁵ targets) in160 μl of complete RPMI medium in a 96 well plate. 20 μl ofphycoerythrin-labeled anti-CD107a Ab (BD Biosciences, 555801) was addedand the plate was incubated at 37° C. for 1 hour before adding GolgiStop (2 ul Golgi Stop in 3 ml RPMI medium, 20 ul/well; BD Biosciences,51-2092KZ) and incubating for another 2.5 hours. Then 5 μl FITC-anti-CD8and 5 ul APC-anti-CD3 were added and incubated at 37° C. for 30 min.After incubation, the samples were washed with FACS buffer and analyzedby flow cytometry.

CFSE Based T Cells Proliferation Assay

Resting CD4 T cells were washed and suspended at a concentration of1×10⁷ cells/ml in

PBS. Then 120 ul CFSE working solution (25 μM CFSE) was added to 1×10⁷cells for 3.5 min at 25° C. The labeling was stopped with 5% FBS (inPBS), washed twice with 5% FBS and cultured in R10 with 10 IU/ml IL2.After overnight culture, the CFSE labeled T cells were electroporatedwith different affinity ErbB2 CAR RNA. Two to four hours afterelectroporation, T cells were suspended at concentration of 1×10⁶/ml inR10 medium (with 10 IU/ml IL2). Tumor or K562 cell lines were irradiatedand suspended at 1×10⁶/mL in R10 medium. Cells were plated at an E:T of1:1 (5×10⁵ effectors: 5×10⁵ targets) in 1 ml of complete RPMI medium ina 48 well plate. T cells were then counted and fed every 2 days from day3. CFSE dilution was monitored by flow cytometry at day 3, day 5 and day7.

Luciferase Based CTL Assay.

Nalm6-CBG tumor cells were generated and employed in a modified versionof a luciferase based CTL assay as follows: Click beetle greenluciferase (CBG) was cloned into the pELNS vector, packaged intolentivirus, transduced into NALM6 tumor cells and sorted for CBGexpression. Resulting Nalm6-CBG cells were washed and resuspended at1×10⁵ cells/ml in R10 medium, and 100 ul of CBG-labeled cells wereincubated with different ratios of T cells (e.g. 30:1, 15:1, etc)overnight at 37° C. 100 μl of the mixture was transferred to a 96 wellwhite luminometerplate, 100 ul of substrate was added and theluminescence was immediately determined.

Mouse Xenograft Studies

Studies were performed as previously described with certainmodifications (Barrett et al., 2011, Human Gene Therapy, 22:1575; andCarpenito et al., 2009, PNAS, 106:336). Briefly, 6-10 week old NOD scidgamma (NSG) mice were injected subcutaneously with 1×10⁶ PC3-CBG tumorscells on the right flank at day 0 and the same mice were givenSK-OV3-CBG tumor cells (5×10⁶ cells/mouse, s.c.) on the left flank atday 5. The mice were treated with T cells via the tail vein at day 23post PC3-CBG tumor inoculation such that both tumors were approximately200 mm³ in volume. Lentivirally transduced T cells were given at 1×10⁷cells/mouse (10M), or 3×10⁶ cells/mouse (3M). RNA electroporated T cellswere given at 5×10⁷ cells/mouse for the 1st treatment, followed by 3treatments at days 26, 30 and 33 in the dose of 1×10⁷ RNA electroporatedT cells/mouse.

Results

Lowering the Affinity of the Anti-ErbB2 scFv Improves the TherapeuticIndex of ErbB2 CAR T Cells In Vitro

A panel of tumor lines with a wide range of ErbB2 expression as measuredby flow cytometry was compiled. SK-OV3 (ovarian cancer), SK-BR3 (breastcancer), BT-474 (breast cancer) over-express ErbB2, while EM-Meso(mesothelioma), MCF7 (breast cancer), 293T (embryonic kidney 293 cell),A549 (lung cancer), 624mel (melanoma), PC3 (prostate cancer), MDA231(breast cancer) express ErbB2 at lower levels and ErbB2 was not detectedin MDA468 (breast cancer). ErbB2 mRNA levels were also measured by realtime PCR and there was a strong correlation between the two techniques.

A panel of ErbB2 CARs was constructed making use scFv derived from thepublished mutations of the parental 4D5 antibody (Carter et al. (1992)Proc Natl Acad Sci USA 89:4285-4289). The sequences encoding the CARsagainst ErbB2 are provided in Table 2.

TABLE 2 Nucleic acid sequences encoding CARs against ErbB2 SEQ CAR IDDesignation Nucleic Acid Sequence NO: 4D5-BZZ atg gac ttc cag gtt cagatc ttt tcg ttc ctg ctg atc agc gcc tct gtt atc atg 40 tcg cgc ggc gacatc cag atg acc cag tcc cct tcc tcc ctc tct gcc tct gtg gga gac cgc gttacc atc aca tgc cga gct tcc cag gac gtg aac aca gcc gtg gcc tgg tac cagcag aag ccc ggg aag gca ccc aaa ctc ctc atc tac tcc gcc tcc ttc cta tacagt ggc gtg cct tcc cga ttc tcc ggc tcc agg agt ggc acg gac ttt acg ctcacc att agt agc ctg cag ccc gaa gac ttc gcg acc tac tat tgt cag caa cactac acg acg cca cca act ttc ggc cag ggt acc aag gtc gag att aag cga accggc agt acc agt ggg tct ggc aag ccc ggc agc ggc gag gga tcc gag gtc cagctg gtc gag tcc ggc ggg ggc ctg gtg cag ccg ggc ggc tcg ctg agg tta tcttgc gcc gcc agt ggc ttc aac atc aag gat act tac atc cac tgg gtg agg caggct ccg ggc aag ggc ctg gaa tgg gtg gct agg atc tac cct act aac ggg tacaca cgc tac gca gat tcg gtg aaa ggc cgc ttc act atc tcc gcc gac acc tcgaag aac act gct tac ctg cag atg aac tcc ctc agg gcc gaa gat act gca gtctac tac tgc tcc cgc tgg ggt ggg gac ggc ttc tac gcc atg gac gtg tgg ggtcag ggc act cta gtt aca gtg tca tcc acc acg acg cca gcg ccg cga cca ccaaca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc cca gag gcg tgccgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac ttc gcc tgt gatatc tac atc tgg gcg ccc ttg gcc ggg act tgt ggg gtc ctt ctc ctg tca ctggtt atc acc ctt tac tgc aaa cgg ggc aga aag aaa ctc ctg tat ata ttc aaacaa cca ttt atg aga cca gta caa act act caa gag gaa gat ggc tgt agc tgccga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctg aga gtg aag ttc agc aggagc gca gac gcc ccc gcg tac aag cag ggc cag aac cag ctc tat aac gag ctcaat cta gga cga aga gag gag tac gac gtt ttg gac aag aga cgt ggc cgg gaccct gag atg ggg gga aag ccg aga agg aag aac cct cag gaa ggc ctg tac aatgaa ctg cag aaa gat aag atg gcg gag gcc tac agt gag att ggg atg aaa ggcgag cgc cgg agg ggc aag ggg cac gat ggc ctt tac cag ggt ctc agt aca gccacc aag gac acc tac gac gcc ctt cac atg cag gcc ctg ccc cct cgc taa4D5-1-BBZ atg gac ttc cag gtt cag atc ttt tcg ttc ctg ctg atc agc gcctct gtt atc atg 41 tcg cgc ggc gac atc cag atg acc cag tcc cct tcc tccctc tct gcc tct gtg gga gac cgc gtt acc atc aca tgc cga gct tcc cag gacgtg aac aca gcc gtg gcc tgg tac cag cag aag ccc ggg aag gca ccc aaa ctcctc atc tac tcc gcc tcc ttc cta gag agt ggc gtg cct tcc cga ttc tcc ggctcc ggc agt ggc acg gac ttt acg ctc acc att agt agc ctg cag ccc gaa gacttc gcg acc tac tat tgt cag caa cac tac acg acg cca cca act ttc ggc cagggt acc aag gtc gag att aag cga acc ggc agt acc agt ggg tct ggc aag cccggc agc ggc gag gga tcc gag gtc cag ctg gtc gag tcc ggc ggg ggc ctg gtgcag ccg ggc ggc tcg ctg agg tta tct tgc gcc gcc agt ggc ttc aac atc aaggat act tac atc cac tgg gtg agg cag gct ccg ggc aag ggc ctg gaa tgg gtggct agg atc tac cct act aac ggg tac aca cgc tac gca gat tcg gtg aaa ggccgc ttc act atc tcc agg gac gac tcg aag aac act ctg tac ctg cag atg aactcc ctc agg gcc gaa gat act gca gtc tac tac tgc gcc cgc tgg ggt ggg gacggc ttc gta gcc atg gac gtg tgg ggt cag ggc act cta gtt aca gtg tca tccacc acg acg cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cagccc ctg tcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cacacg agg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc gggact tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggcaga aag aaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa actact caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga ggatgt gaa ctg aga atg gac ttc cag gtt cag atc ttt tcg ttc ctg ctg atc agcgcc tct gtt atc atg tcg cgc ggc gac atc cag atg acc cag tcc cct tcc tccctc tct gcc tct gtg gga gac cgc gtt acc atc aca tgc cga gct tcc cag gacgtg aac aca gcc gtg gcc tgg tac cag cag aag ccc ggg aag gca ccc aaa ctcctc atc tac tcc gcc tcc ttc cta gag agt ggc gtg cct tcc cga ttc tcc ggctcc ggc agt ggc acg gac ttt acg ctc acc att agt agc ctg cag ccc gaa gacttc gcg acc tac tat tgt cag caa cac tac acg acg cca cca act ttc ggc cagggt acc aag gtc gag att aag cga acc ggc agt acc agt ggg tct ggc aag cccggc agc ggc gag gga tcc gag gtc cag ctg gtc gag tcc ggc ggg ggc ctg gtgcag ccg ggc ggc tcg ctg agg tta tct tgc gcc gcc agt ggc ttc aac atc aaggat act tac atc cac tgg gtg agg cag gct ccg ggc aag ggc ctg gaa tgg gtggct agg atc tac cct act aac ggg tac aca cgc tac gca gat tcg gtg aaa ggccgc ttc act atc tcc agg gac gac tcg aag aac act ctg tac ctg cag atg aactcc ctc agg gcc gaa gat act gca gtc tac tac tgc gcc cgc tgg ggt ggg gacggc ttc gta gcc atg gac gtg tgg ggt cag ggc act cta gtt aca gtg tca tccgtg aag ttc agc agg agc gca gac gcc ccc gcg tac aag cag ggc cag aac cagctc tat aac gag ctc aat cta gga cga aga gag gag tac gac gtt ttg gac aagaga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aac cct caggaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agt gagatt ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt tac cagggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atg cag gcc ctgccc cct cgc taa 4D5-3-BBZ acc acg acg cca gcg ccg cga cca cca aca ccggcg ccc acc atc gcg tcg 42 cag ccc ctg tcc ctg cgc cca gag gcg tgc cggcca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac ttc gcc tgt gat atctac atc tgg gcg ccc ttg gcc ggg act tgt ggg gtc ctt ctc ctg tca ctg gttatc acc ctt tac tgc aaa cgg ggc aga aag aaa ctc ctg tat ata ttc aaa caacca ttt atg aga cca gta caa act act caa gag gaa gat ggc tgt agc tgc cgattt cca gaa gaa gaa atg gac ttc cag gtt cag atc ttt tcg ttc ctg ctg atcagc gcc tct gtt atc atg tcg cgc ggc gac atc cag atg acc cag tcc cct tcctcc ctc tct gcc tct gtg gga gac cgc gtt acc atc aca tgc cga gct tcc caggac gtg aac aca gcc gtg gcc tgg tac cag cag aag ccc ggg aag gca ccc aaactc ctc atc tac tcc gcc tcc ttc cta gag agt ggc gtg cct tcc cga ttc tccggc tcc ggc agt ggc acg gac ttt acg ctc acc att agt agc ctg cag ccc gaagac ttc gcg acc tac tat tgt cag caa cac tac acg acg cca cca act ttc ggccag ggt acc aag gtc gag att aag cga acc ggc agt acc agt ggg tct ggc aagccc ggc agc ggc gag gga tcc gag gtc cag ctg gtc gag tcc ggc ggg ggc ctggtg cag ccg ggc ggc tcg ctg agg tta tct tgc gcc gcc agt ggc ttc aac atcaag gat act tac atc cac tgg gtg agg cag gct ccg ggc aag ggc ctg gaa tgggtg gct agg atc tac cct act aac ggg tac aca cgc tac gca gat tcg gtg aaaggc cgc ttc act atc tcc gcc gac acc tcg aag aac act gct tac ctg cag atgaac tcc ctc agg gcc gaa gat act gca gtc tac tac tgc tcc cgc tgg ggt ggggac ggc ttc gta gcc atg gac gtg tgg ggt cag ggc act cta gtt aca gtg tcatcc gaa gga gga tgt gaa ctg aga gtg aag ttc agc agg agc gca gac gcc cccgcg tac aag cag ggc cag aac cag ctc tat aac gag ctc aat cta gga cga agagag gag tac gac gtt ttg gac aag aga cgt ggc cgg gac cct gag atg ggg ggaaag ccg aga agg aag aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gataag atg gcg gag gcc tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggcaag ggg cac gat ggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc tacgac gcc ctt cac atg cag gcc ctg ccc cct cgc taa 4D5-5-BBZ atg gac ttccag gtt cag atc ttt tcg ttc ctg ctg atc agc gcc tct gtt atc atg 43 tcgcgc ggc gac atc cag atg acc cag tcc cct tcc tcc ctc tct gcc tct gtg ggagac cgc gtt acc atc aca tgc cga gct tcc cag gac gtg aac aca gcc gtg gcctgg tac cag cag aag ccc ggg aag gca ccc aaa ctc ctc atc tac tcc gcc tccttc cta gag agt ggc gtg cct tcc cga ttc tcc ggc tcc agg agt ggc acg gacttt acg ctc acc att agt agc ctg cag ccc gaa gac ttc gcg acc tac tat tgtcag caa cac tac acg acg cca cca act ttc ggc cag ggt acc aag gtc gag attaag cga acc ggc agt acc agt ggg tct ggc aag ccc ggc agc ggc gag gga tccgag gtc cag ctg gtc gag tcc ggc ggg ggc ctg gtg cag ccg ggc ggc tcg ctgagg tta tct tgc gcc gcc agt ggc ttc aac atc aag gat act tac atc cac tgggtg agg cag gct ccg ggc aag ggc ctg gaa tgg gtg gct agg atc tac cct actaac ggg tac aca cgc tac gca gat tcg gtg aaa ggc cgc ttc act atc tcc gccgac acc tcg aag aac act gct tac ctg cag atg aac tcc ctc agg gcc gaa gatact gca gtc tac tac tgc tcc cgc tgg ggt ggg gac ggc ttc gta gcc atg gacgtg tgg ggt cag ggc act cta gtt aca gtg tca tcc acc acg acg cca gcg ccgcga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc ccagag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac ttcgcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg act tgt ggg gtc ctt ctcctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga aag aaa ctc ctg tatata ttc aaa caa cca ttt atg aga cca gta caa act act caa gag gaa gat ggctgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctg aga gtg aagttc agc agg agc gca gac gcc ccc gcg tac aag cag ggc cag aac cag ctc tataac gag ctc aat cta gga cga aga gag gag tac gac gtt ttg gac aag aga cgtggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aac cct cag gaa ggcctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agt gag att gggatg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt tac cag ggt ctcagt aca gcc acc aag gac acc tac gac gcc ctt cac atg cag gcc ctg ccc cctcgc taa 4D5-7-BBZ atg gac ttc cag gtt cag atc ttt tcg ttc ctg ctg atcagc gcc tct gtt atc atg 44 tcg cgc ggc gac atc cag atg acc cag tcc ccttcc tcc ctc tct gcc tct gtg gga gac cgc gtt acc atc aca tgc cga gct tcccag gac gtg aac aca gcc gtg gcc tgg tac cag cag aag ccc ggg aag gca cccaaa ctc ctc atc tac tcc gcc tcc ttc cta gag agt ggc gtg cct tcc cga ttctcc ggc tcc agg agt ggc acg gac ttt acg ctc acc att agt agc ctg cag cccgaa gac ttc gcg acc tac tat tgt cag caa cac tac acg acg cca cca act ttcggc cag ggt acc aag gtc gag att aag cga acc ggc agt acc agt ggg tct ggcaag ccc ggc agc ggc gag gga tcc gag gtc cag ctg gtc gag tcc ggc ggg ggcctg gtg cag ccg ggc ggc tcg ctg agg tta tct tgc gcc gcc agt ggc ttc aacatc aag gat act tac atc cac tgg gtg agg cag gct ccg ggc aag ggc ctg gaatgg gtg gct agg atc tac cct act aac ggg tac aca cgc tac gca gat tcg gtgaaa ggc cgc ttc act atc tcc gcc gac acc tcg aag aac act gct tac ctg cagatg aac tcc ctc agg gcc gaa gat act gca gtc tac acc acg acg cca gcg ccgcga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc ccagag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac ttcgcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg act tgt ggg gtc ctt ctcctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga aag aaa ctc ctg tatata ttc aaa caa cca ttt atg aga cca gta caa act act caa gag gaa gat ggctgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctg aga gtg aagttc agc agg agc gca gac gcc ccc gcg tac aag cag ggc cag aac cag ctc tataac gag ctc aat cta gga cga aga gag gag tac gac gtt ttg gac aag aga cgtggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aac cct cag gaa ggcctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agt gag att gggatg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt tac cag ggt ctcagt aca gcc acc aag gac acc tac gac gcc ctt cac atg cag gcc ctg ccc cctcgc taa

The monovalent affinities of the ErbB2 scFvs varied by approximately 3orders of magnitude (Table 3), in contrast to the corresponding mutantantibodies that retained binding affinities within 10-fold of each other(Carter, P., et al. 1992).

TABLE 3 Comparison of measured affinities of the wild type 4D5 andmutated antibody with the corresponding scFv Antibody scFv SampleMutation KD (nM) KD (nM) 4D5 Wild Type 0.3 0.58 4D5-7 1 in CDR2 0.62 3.24D5-5 1 in CDR3, 1 in CDR2 1.1 1119 4D5-3 1 in framework, 1 in CDR3, 1in CDR2 4.4 3910

CARs were constructed by linking the various scFv to the CD8 alpha hingeand transmembrane domain followed by the 4-1BB and CD3ζ intracellularsignaling domains. The CARs were expressed by lentiviral vectortechnology or by cloning into an RNA-based vector (Zhao et al., 2010,Cancer Res, 70:9053). After production of mRNA by in vitro transcriptionand electroporation into T cells, the surface expression of the panel ofaffinity-modified ErbB2 RNA CARs was similar. To compare recognitionthresholds, the panel of ErbB2 CAR T cells was stimulated with ErbB2high expressing (SK-BR3, SK-OV3 and BT-474) or low expressing tumor celllines (MCF7, 293T, A549, 624Mel, PC3, MDA231 and MDA468) and T cellactivation was assessed by upregulation of CD137 (4-1BB;), secretion ofIFN-γ and IL-2 and induction of surface CD107a expression. T cellsexpressing a CD19-specific CAR served as control for allogeneicreactivity. Lower affinity CAR T cells (4D5-5 and 4D5-3) were stronglyreactive to tumors with amplified ErbB2 expression and exhibitedundetectable or low reactivity to the tumor lines that expressed ErbB2at lower levels. In contrast, higher affinity CAR T cells (4D5 and4D5-7) showed strong reactivity to tumor lines expressing high and lowlevels of ErbB2, as evidenced by CD137 up-regulation, cytokine secretionand CD107a translocation. These results were extended by assayingadditional ErbB2-expressing cell lines. Interestingly, higher affinityCAR T cells secreted greater levels of IFN-γ and IL-2 when exposed totargets expressing low levels of ErbB2, while lower affinity CAR T cellssecreted more cytokines when exposed to cells expressing high levels oftarget. As expected, the CD19-BBζ CAR was not reactive againstErbB2-expressing cell lines. In summary, higher affinity 4D5-BBζ T cellsrecognized all the ErbB2 expressing lines tested, whereas CARs withlower affinity scFvs, 4D5-5-BBζ or 4D5-3-BBζ, were highly reactive toall tumor lines with overexpressed ErbB2, but displayed negligiblereactivity to cell lines expressing low or undetectable levels of ErbB2.

ErbB2 CARS with Lower Affinity scFvs Discriminate Between Tumor CellsExpressing Low and High Levels of ErbB2.

To exclude any tumor-specific effects that might contribute to the aboveresults, the activity of the panel of ErbB2-BBζ CAR T cells was assayedagainst a single tumor line expressing varying levels of ErbB2 (K562cells electroporated with varying amounts of ErbB2 RNA). In agreement,it was observed that T cells expressing higher affinity scFvs (4D5 and4D5-7) recognized K562 cells electroporated with ErbB2 RNA at doses aslow as 0.001 μg, which is 100 fold lower than the flow cytometricallydetectable level of 0.1 μg mRNA. In contrast the CARs with loweraffinity scFvs (4D5-5 and 4D5-3) only recognized K562 electroporatedErbB2 RNA at doses of 0.5 μg (4D5-5; 10) or higher, indicating that CART cell sensitivity was decreased by 2000-(4D5-3) to 500-fold (4D5-5)compared to the high affinity 4D5 CAR T cells. Moreover, the antigendose associated reactivity observed with lower affinity ErbB2 CARs(4D5-5 and 4D5-3), was confirmed by performing a CFSE-basedproliferation assay. Interestingly, decreasing the CAR RNA dose 5 fold(from 10 μg RNA/100 μl T cells to 2 μg RNA/100 μl T cells), furtherincreased the antigen recognition threshold of the T cells with lowerand high affinity CARs as assessed by cytokine secretion, suggestingthat fine tuning of CAR density on the surface of the T cells is animportant variable, or that doses above 2 μg of mRNA may have sometoxicity on overall T cell activity.

A luciferase based cytolytic T cell (CTL) assay was used to determinewhether T cells with affinity decreased CARs could maintain potentkilling activity against ErbB2 over expressing targets while sparingcells expressing lower ErbB2 levels. When Nalm6 target cells weretransfected with 10 μg ErbB2 RNA, T cells with either higher or loweraffinity ErbB2 CARs effectively lysed target cells. CARs with higheraffinity scFv (4D5 and 4D5-7) exhibit potent lytic activity againsttarget cells transfected with 1 μg ErbB2 RNA, but lower affinity scFvs(4D5-5 and 4D5-3) showed decreased killing activity. Finally, only CARswith higher affinity scFvs were able to kill target cells expressingvery low amounts of target after electroporation with 0.1 μg ErbB2 RNA.Since Nalm6 is a CD19 positive cell line, CART19 maintained cytolyticactivity independent of levels of transfected ErbB2 RNA. These dataindicate that that fine-tuning the affinity of ErbB2 CAR T cellsenhances discrimination of ErbB2 over-expressing tumor from tumor cellsthat have low or undetectable levels of ErbB2 expression.

Affinity Decreased ErbB2 CAR T Cells Fail to Recognize PhysiologicalLevels of ErbB2

Given the previous serious adverse event which occurred uponadministration of the high affinity ErbB2 CAR that incorporated the scFvfrom the parental 4D5 trastuzumab antibody (Morgan et al., 2010, MolTherapy, 18:843), it is of paramount importance to evaluate potentialreactivity of the reduced affinity ErbB2 CAR T cells to physiologicallevels of ErbB2 expression. To address this, seven primary cell linesisolated from different organs were tested for ErbB2 expression. Most ofthe primary lines had detectable levels of surface ErbB2, with theneural progenitor line expressing the highest levels of ErbB2. T cellsexpressing the high affinity 4D5 CAR were strongly reactive to allprimary lines tested, as evidenced by levels of CD107a up-regulation.However, T cells expressing the affinity decreased ErbB2 CARs 4D5-5 and4D5-3 exhibited no reactivity to the primary lines with the exception ofweak reactivity to the neural progenitor line. These results wereconfirmed by analysis of a larger panel of cell lines that had low orundetectable levels of ErbB2 by flow cytometry.

Comparable Effects with Affinity-Tuned ErbB2 CARs Expressed UsingLentiviral Transduction or RNA Electroporation

To establish comparability between T cells permanently expressing CARsby lentiviral transduction with mRNA electroporated CAR T cells, thepanel of affinity-tuned CARs was expressed in T cells from the samenormal donor using either lentiviral transduction or mRNAelectroporation. T cells were stimulated with tumor cell lines, or K562cells, expressing varying amounts of ErbB2. CAR T cell recognition andactivation was monitored by CD107a upregulation, CD137 upregulation andIFN-γ secretion. In agreement with the previous ErbB2 mRNA CAR T cellresults, T cells that constitutively expressed high affinity CARs showedstrong reactivity to all cell lines expressing ErbB2; no correlation wasobserved between antigen expression levels and T cell-activity. Incontrast, T cells with low affinity CARs expressed by lentiviraltechnology demonstrated a robust correlation between target antigenexpression and activation. These results confirm that the sensitivity ofErbB2 antigen recognition is dependent on scFv affinity using both mRNAelectroporated and lentiviral transduced CAR T cells.

Affinity Decreased ErbB2 CAR T Cells Eliminate Tumor In Vivo and IgnoreTissues Expressing Physiological Levels of ErbB2

To extend the above in vitro results, a series of experiments wereconducted in NSG mice with advanced vascularized tumor xenografts. Thehuman ovarian cancer cell line SK-OV3 was selected as a representativeErbB2 over-expressing tumor and PC3, a human prostate cancer line, waschosen to model normal tissue ErbB2 levels. The antitumor efficacy ofErbB2 CAR T cells expressing either the high affinity 4D5 scFv or thelow affinity 45D-5 scFv in NSG mice was compared with day 18 establishedflank SK-OV3 tumors. Serial bioluminescence imaging revealed that boththe high and low affinity CAR T cells resulted in the rapid eliminationof the tumors.

To further evaluate the therapeutic index of the low affinity ErbB2 CART cells in vivo, a mouse model was designed to simultaneously comparethe efficacy and normal tissue toxicity of the high affinity (4D5:BBζ)and low affinity (4D5-5:BBζ) ErbB2 CARs. SK-OV3 and PC3 tumor cell lineswere injected subcutaneously into opposite flanks of the same NSG mouseand T cells were administered when tumor volumes reached approximately200 mm³. Mice were injected (i.v.) with either 3×10⁶ or 1×10⁷ CAR Tcells on day 22 and serial bioluminescence imaging and tumor sizeassessments were conducted. Mice treated with either dose of the CAR Tcells exhibited nearly complete regression of the ErbB2 overexpressingSK-OV3 tumor. In addition, almost complete regression of the PC3 tumorexpressing ErbB2 at low levels on the opposite flank was also seen forthe mice treated with high affinity 4D5-based CAR T cells. In contrast,the progressive tumor growth of PC3 was observed in the mice treatedwith low affinity 4D5-5-based CAR T cells, indicating that whereas thelower affinity CAR T cells were efficacious against ErbB2 overexpressingtumor, they show limited or no detectable reactivity against cellsexpressing ErbB2 at physiological levels. Moreover, the selective tumorelimination was observed in mice treated at both high and low doses ofCAR T cells. The above effects were not due to allorecognition becauseprogressive tumor growth of both tumors was observed in mice treatedwith mock transduced T cells.

Affinity-Tuning of scFv Increases the Therapeutic Index of EGFR CAR TCells

To test the broader applicability the strategy to fine tune the affinityof the scFv, we evaluated a panel of EGFR CARs. EGFR:BBζ CARs wereconstructed from scFvs derived from the parental human anti-EGFRantibody C10 (Heitner et al., 2001, J Immunol Methods, 248:17-30. Thenucleic acid sequences encoding the EGFR CARs are provided in Table 4.

TABLE 4 Nucleic Acid Sequences of Exemplary EGFR CARs CAR SEQ IDdesignation Nucleic Acid Sequence NO: C10-BBZ atg ggt tgg tcg tgc attatc ctc ttc ctc gtc gca acc gct acc ggc gtt cac 45 tcg gat tac aag gatgac gac gac aaa gag gta cag ctg gtg cag agc ggg gcc gag gtt aag aag cccggg tct tcc gta aag gtg tcc tgc aag gcc tcg ggg ggc aca ttc tca tcg tacgca ata tcg tgg gtg cgg cag gcc ccc ggg cag ggg ctg gaa tgg atg ggc ggaatt atc cca atc ttc ggg acc gcc aac tat gcc cag aag ttt cag ggt cgt gtgacc att act gcc gac gag tcc acc agt acg gcc tac atg gag ctg agt agt ctgcgt agc gag gat act gcc gtt tat tat tgc gcc cgg gaa gag gga ccg tac tgctcg tcg acc tca tgt tac ggc gcc ttc gac atc tgg ggc caa ggc acc ctg gtgacg gtg tcc tcc ggt ggt ggc gga agt ggc ggc ggg ggg tcc ggc ggg ggc ggttca cag tcc gtc ctg acc cag gat ccc gcg gtg tcg gtc gcg ctg ggt cag acagta aag ata aca tgc cag ggc gat tct ctg cgc agt tat ttc gcc tcg tgg taccag cag aaa ccc ggc cag gct cct acc ctt gtt atg tac gcg cgc aat gac agaccc gcg ggc gtg ccc gac cgc ttc tcc ggc tca aag agc ggg acc tcc gcc tccctg gcc atc tcc ggg ctc cag tct gag gat gag gcc gat tac tac tgc gct gcttgg gac gac tcc ctc aat ggc tat ctg ttt ggc gca ggc aca aag ctg acc gtgctc acc acg acg cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcgcag ccc ctg tcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtgcac acg agg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gccggg act tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cggggc aga aag aaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caaact act caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa ggagga tgt gaa ctg aga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac aagcag ggc cag aac cag ctc tat aac gag ctc aat cta gga cga aga gag gag tacgac gtt ttg gac aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg agaagg aag aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcggag gcc tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cacgat ggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc cttcac atg cag gcc ctg ccc cct cgc taa 2224-BBZ atg ggt tgg tcg tgc att atcctc ttc ctc gtc gca acc gct acc ggc gtt cac 46 tcg gat tac aag gat gacgac gac aaa gag gta cag ctg gtg cag agc ggg gcc gag gtt aag aag ccc gggtct tcc gta aag gtg tcc tgc aag gcc tcg ggg ggc aca ttc tca tcg tac gcaata ggt tgg gtg cgg cag gcc ccc ggg cag ggg ctg gaa tgg atg ggc gga attatc cca atc ttc ggg atc gcc aac tat gcc cag aag ttt cag ggt cgt gtg accatt act gcc gac gag tcc acc agt agt gcc tac atg gag ctg agt agt ctg cgtagc gag gat act gcc gtt tat tat tgc gcc cgg gaa gag gga ccg tac tgc tcgtcg acc tca tgt tac gca gcc ttc gac atc tgg ggc caa ggc acc ctg gtg acggtg tcc tcc ggt ggt ggc gga agt ggc ggc ggg ggg tcc ggc ggg ggc ggt tcacag tcc gtc ctg acc cag gat ccc gcg gtg tcg gtc gcg ctg ggt cag aca gtaaag ata aca tgc cag ggc gat tct ctg cgc agt tat ttc gcc tcg tgg tac cagcag aaa ccc ggc cag gct cct acc ctt gtt atg tac gcg cgc aat gac aga cccgcg ggc gtg ccc gac cgc ttc tcc ggc tca aag agc ggg acc tcc gcc tcc ctggcc atc tcc ggg ctc cag ccc gag gat gag gcc gat tac tac tgc gct gct tgggac gac tcc ctc aat ggc tat ctg ttt ggc gca ggc aca aag ctg acc gtg ctcacc acg acg cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cagccc ctg tcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cacacg agg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc gggact tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggcaga aag aaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa actact caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga ggatgt gaa ctg aga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac aag cagggc cag aac cag ctc tat aac gag ctc aat cta gga cga aga gag gag tac gacgtt ttg gac aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga aggaag aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gaggcc tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gatggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cacatg cag gcc ctg ccc cct cgc taa 3524-BBZ atg ggt tgg tcg tgc att atc ctcttc ctc gtc gca acc gct acc ggc gtt cac 47 tcg gat tac aag gat gac gacgac aaa gag gta cag ctg gtg cag agc ggg gcc gag gtt aag aag ccc ggg tcttcc gta aag gtg tcc tgc aag gcc tcg ggg ggc aca ttc tca tcg tac gca atatcg tgg gtg cgg cag gcc ccc ggg cag ggg ctg gaa tgg gtc ggc gga att atccca atc ttc ggg acc gcc aac tat gcc cag aag ttt cag ggt cgt gtg aag attact gcc gac gag tcc gca agt acg gcc tac atg gag ctg agt agt ctg cgt agcgag gat act gcc gtt tat tat tgc gcc cgg gaa gag gga ccg tac tgc tcg tcgacc tca tgt tac gca gcc ttc gac atc tgg ggc caa ggc acc ctg gtg acg gtgtcc tcc ggt ggt ggc gga agt ggc ggc ggg ggg tcc ggc ggg ggc ggt tca cagtcc gtc ctg acc cag gat ccc gcg gtg tcg gtc gcg ctg ggt cag aca gta aagata aca tgc cag ggc gat tct ctg cgc agt tat ctg gcc tcg tgg tac cag cagaaa ccc ggc cag gct cct acc ctt gtt acc tac gcg cgc aat gac aga ccc gcgggc gtg ccc gac cgc ttc tcc ggc tca aag agc ggg acc tcc gcc tcc ctg gccatc tcc ggg ctc cag tct gag gat gag gcc gat tac tac tgc gct gct tgg gacgac tcc ctc aat ggc tat ctg ttt ggc gca ggc aca aag ctg acc gtg ctc accacg acg cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cag cccctg tcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acgagg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg acttgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc agaaag aaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa act actcaa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgtgaa ctg aga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac aag cag ggccag aac cag ctc tat aac gag ctc aat cta gga cga aga gag gag tac gac gttttg gac aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aagaac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcctac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggcctt tac cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atgcag gcc ctg ccc cct cgc taa P3-5BBZ atg ggt tgg tcg tgc att atc ctc ttcctc gtc gca acc gct acc ggc gtt cac 48 tcg gat tac aag gat gac gac gacaaa gag gta cag ctg gtg cag agc ggg gcc gag gtt aag aag ccc ggg tct tccgta aag gtg tcc tgc aag gcc tcg ggg ggc aca ttc tca tcg tac gca ata tcgtgg gtg cgg cag gcc ccc ggg cag ggg ctg gaa tgg gtc ggc gga att atc ccaatc ttc ggg acc gcc aac tat gcc cag aag ttt cag ggt cgt gtg aag att actgcc gac gag tcc gca agt acg gcc tac atg gag ctg agt agt ctg cgt agc gaggat act gcc gtt tat tat tgc gcc cgg gaa gag gga ccg tac tgc tcg tcg acctca tgt tac ggc gcc ttc gac atc tgg ggc caa ggc acc ctg gtg acg gtg tcctcc ggt ggt ggc gga agt ggc ggc ggg ggg tcc ggc ggg ggc ggt tca cag tccgtc ctg acc cag gat ccc gcg gtg tcg gtc gcg ctg ggt cag aca gta aag ataaca tgc cag ggc gat tct ctg cgc agt tat ctg gcc tcg tgg tac cag cag aaaccc ggc cag gct cct acc ctt gtt acc tac gcg cgc aat gac aga ccc gcg ggcgtg ccc gac cgc ttc tcc ggc tca aag agc ggg acc tcc gcc tcc ctg gcc atctcc ggg ctc cag tct gag gat gag gcc gat tac tac tgc gct gct tgg gac gactcc ctc aat ggc tat ctg ttt ggc gca ggc aca aag ctg acc gtg ctc acc acgacg cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctgtcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg aggggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg act tgtggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga aagaaa ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa act act caagag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgt gaactg aga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac aag cag ggc cagaac cag ctc tat aac gag ctc aat cta gga cga aga gag gag tac gac gtt ttggac aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aaccct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tacagt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctttac cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atg caggcc ctg ccc cct cgc taa P2-4BBZ atg ggt tgg tcg tgc att atc ctc ttc ctcgtc gca acc gct acc ggc gtt cac 49 tcg gat tac aag gat gac gac gac aaagag gta cag ctg gtg cag agc ggg gcc gag gtt aag aag ccc ggg tct tcc gtaaag gtg tcc tgc aag gcc tcg ggg ggc aca ttc tca tcg tac gca ata tcg tgggtg cgg cag gcc ccc ggg cag ggg ctg gaa tgg atg ggc gga att atc cca atcttc ggg acc gcc aac tat gcc cag aag ttt cag ggt cgt gtg acc att act gccgac gag tcc acc agt acg gcc tac atg gag ctg agt agt ctg cgt agc gag gatact gcc gtt tat tat tgc gcc cgg gaa gag gga ccg tac tgc tcg tcg acc tcatgt tac gca gcc ttc gac atc tgg ggc caa ggc acc ctg gtg acg gtg tcc tccggt ggt ggc gga agt ggc ggc ggg ggg tcc ggc ggg ggc ggt tca cag tcc gtcctg acc cag gat ccc gcg gca tcg gtc gcg ctg ggt cag aca gta aag ata acatgc cag ggc gat tct ctg cgc agt tat ttc gcc tcg tgg tac cag cag aaa cccggc cag gct cct acc ctt gtt atg tac gcg cgc aat gac aga ccc gcg ggc gtgccc gac cgc ttc tcc ggc tca aag agc ggg acc tcc gcc tcc ctg gcc atc tccggg ctc cag tct gag gat gag gcc gat tac tac tgc gct gct tgg gac gac tccctc aat ggc tat ctg ttt ggc gca ggc aca aag ctg acc gtg ctc acc acg acgcca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tccctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg gggctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg act tgt ggggtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga aag aaactc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa act act caa gaggaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctgaga gtg aag ttc agc agg agc gca gac gcc ccc gcg tac aag cag ggc cag aaccag ctc tat aac gag ctc aat cta gga cga aga gag gag tac gac gtt ttg gacaag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aac cctcag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agtgag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt taccag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atg cag gccctg ccc cct cgc taa

The monovalent affinities of the panel of EGFR-specific scFvs variedover a range of approximately 300-fold (Zhoe et al., 2007, J Mol Biol,371:934). The 2224, P2-4, P3-5 and C10 scFvs were cloned into anRNA-based vector and in vitro transcribed for T cell mRNAelectroporation. Levels of CAR surface expression were assayed and foundto be similar among the EGFR constructs. To compare reactivities of thepanel of EGFR CARs, CAR T cells were stimulated with EGFR-expressingtumor cell lines that have a broad range of EGFR expression at the cellsurface. CAR T cell activation was evaluated by levels of CD107aup-regulation. Higher affinity EGFR CARs (2224:BBζ and P2-4:BBζ)responded to all EGFR positive tumor lines (MDA468, MDA231 and SK-OV3)regardless of EGFR expression levels. However, the reactivity exhibitedby lower affinity EGFR CARs (P3-5.BBZ and C10.BBZ) againstEGFR-expressing tumor lines did correlate with the levels of EGFRexpression. Furthermore, lower affinity EGFR CARs displayed more potentreactivity to the EGFR overexpressing tumor, MDA468, than the higheraffinity EGFR CARs, while provoking a much weaker response to EGFR lowexpressing cells. None of the EGFR CAR T cells reacted to the EGFRnegative tumor line K562.

To confirm that the level of response was related to scFv affinity andthe level of EGFR expression, and to exclude tumor-specific effects, thepanel of EGFR CAR T cells was co-cultured with K562 cells expressingvarying levels of EGFR after electroporation with EGFR mRNA. The higheraffinity EGFR CARs did not discriminate between target cells withdifferent levels of EGFR expression. For example, T cells expressing CAR2224 responded equally well to K562 cells electroporated with a 200-folddifference in EGFR mRNA (0.1 μg to 20 μg). However in agreement with theabove ErbB2 CAR results, the lower affinity EGFR CARs (P3-5 and C10)exhibited a high correlation between T cell responses and EGFRexpression levels.

To confirm the increased safety profile of the lower affinity EGFR CARs,we tested the reactivities of EGFR CARs against primary cells derivedfrom different organs. Five primary cell lines and five tumor cell lineswere tested for both surface levels of EGFR and ability to trigger CAR Tcell reactivity. Three of the primary cell lines examined expressdetectable levels of EGFR and two did not (pulmonary artery smoothmuscle and PBMC). Two of the tumor cell lines (MCF7 and Raji) did notexpress detectable EGFR on the cell surface. Comparing EGFR CAR T cellsto CD19 CAR T cells, T cells with higher EGFR affinity CARs (2224 andP2-4) reacted to all the primary lines tested and all of the tumorsexcept Raji. However, T cells with the affinity decreased EGFR CAR Tcells P3-5 and C10 were not reactive to any of the five primary cellstested. CD19 specific CAR T cells reacted to the CD19+ line Raji, and toPBMCs, presumably to the B cells in PBMC, but did not respond to any ofthe tumor lines or other primary cell lines. These data demonstrate thataffinity tuning of scFv can increase the therapeutic index for CAR Tcells that target either ErbB2 or EGFR.

Example 3: Optimizing CAR Therapy with Administration of ExogenousCytokines

Cytokines have important functions related to T cell expansion,differentiation, survival and homeostasis. One of the most importantcytokine families for clinical use is the common γ-chain (γ_(c)) familycytokines, which includes interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15and IL-21 (Liao et al., 2013, Immunity, 38:13-25). IL-2 has been widelystudied as an immunotherapeutic agent for cancer. The supplement of IL-2enhanced the antitumor ability of anti-CD19 CAR-T cells in the clinicaltrials (Xu et al., 2013, Lymphoma, 54:255-60). However, theadministration of IL-2 is limited by side effects and a propensity forexpansion of regulatory T cells and the effect of activated induced celldeath (AICD) (Malek et al., 2010, Immunity, 33:153-65; and Lenardo etal., 1999, Annu Rev Immunol, 17:221-53). IL-7, IL-15, and IL-21 each canenhance the effectiveness of adoptive immunotherapies and seems to beless toxicity compared with IL-2 (Alves et al., 2007, Immunol Lett,108:113-20). Despite extensive preclinical and clinical studies on therole of the above cytokines, multi-parameter comparative studies on theroles of various exogenous γ_(c) cytokines on CAR-T cell adoptivetherapy are lacking.

Besides γ-chain cytokines, IL-18 is another immunostimulatory cytokineregulating immune responses, which enhances the production of IFN-γ by Tcells and augments the cytolytic activity of CTLs (Srivastava et al.,2010, Curr Med Chem, 17:3353-7). Administration of IL-18 is safe andwell tolerated, even when the dose reaching as high as 1000 μg/kg(Robertson et al., 2006, Clin Cancer Res, 12:4265-73). Therefore, IL-18could be another candidate used to boost the antitumor of CAR-T cells.

To further enhance the efficacy of adoptive therapy with CAR engineeredT (CAR-T) cells, optimization of CAR therapy with administration ofexogenous cytokines was examined. To compare the roles of differentcytokines administrated exogenously during CAR-T cell immunotherapy andfind the optimal cytokine for clinical use, the in vivo antitumorability of CAR-T cells was tested using ovarian cancer animal models.

The following materials and methods were used in the experimentsdescribed in this example.

CAR Construction and Lentivirus Preparation

The pELNS-C4-27z CAR vector was constructed as described previously(manuscript under review), Briefly, the pHEN2 plasmid containing theanti-FRα C4/AFRA4 scFv was used as a template for PCR amplification ofC4 fragment using the primers of 5′-ataggatcccagctggtggagtctgggggaggc-3′(SEQ ID NO: 50) and 5′-atagctagcacctaggacggtcagcttggtccc-3′ (SEQ ID NO:51) (BamHI and NheI were underlined). The PCR product and the thirdgeneration self-inactivating lentiviral expression vectors pELNS weredigested with BamHI and NheI. The digested PCR products were theninserted into the pELNS vector containing CD27-CD3z T-cell signalingdomain in which transgene expression is driven by the elongationfactor-1α (EF-1α) promoter.

High-titer replication-defective lentivirus was generated bytransfection of human embryonic kidney cell line 293T (293T) cells withfour plasmids (pVSV-G, pRSV.REV, pMDLg/p.RRE and pELNS-C4-27z CAR) byusing Express In (Open Biosystems) as described previously (manuscriptunder review). Supernatants were collected and filtered at 24 h and 48 hafter transfection. The media was concentrated by ultracentrifugation.Alternatively, a single collection was done 30 hr after media change.Virus containing media was alternatively used unconcentrated orconcentrated by Lenti-X concentrator (Clontech, Cat#631232). The virustiters were determined based on the transduction efficiency oflentivirus to SupT1 cells by using limiting dilution method.

T Cells and Cell Lines

Peripheral blood lymphocytes were obtained from healthy donors afterinformed consent under a protocol approved by University InstitutionalReview Board at the University of

Pennsylvania. The primary T cells were purchased from the HumanImmunology Core after purified by negative selection. T cells werecultured in complete media (RPMI 1640 supplemented with 10% FBS, 100U/mL penicillin, 100 μg/mL streptomycin sulfate) and stimulated withanti-CD3 and anti-CD28 mAbs-coated beads (Invitrogen) at a ratio of 1:1following the instruction. Twenty-four hours after activation, cellswere transduced with lentivirus at MOI of 5. Indicated cytokines wereadded to the transduced T cells from the next day with a finalconcentration of 10 ng/mL. The cytokines were replaced every 3 days.

The 293T cell used for lentivirus packaging and the SupT1 cell used forlentiviral titration were obtained from ATCC. The established ovariancancer cell lines SKOV3 (FRα+) and C30 (FRα−) was used as target cellfor cytokine-secreting and cytotoxicity assay. For bioluminescenceassays, SKOV3 was transduced with lentivirus to express fireflyluciferase (fLuc).

Flow Cytometric Analysis and Cell Sorting

Flow cytometry was performed on a BD FACSCanto. Anti-human CD45 (HI30),CD3 (HIT3a), CD8 (HIT8a), CD45RA (HI100), CD62L (DREG-56), CCR7(G043H7), IL-7Rα (A019D5), CD27 (M-T271), CD28 (CD28.2), CD95 (DX2),TNF-α (MAb11), IFN-γ (4S.B3), IL-2 (MQ1-17H12), perforin (B-D48),granzym-B (GB11) were obtained from Biolegend. Biotin-SP-conjugatedrabbit anti-human IgG (H+L) was purchased from Jackson Immunoresearchand APC conjugated streptavidin was purchased from Biolegand. Anti-humanBcl-xl (7B2.5) was purchased from SouhernBiotech. Apoptosis kit andTruCount tubes were obtained from BD Bioscience. For peripheral blood Tcell count, blood was obtained via retro-orbital bleeding and stainedfor the presence of human CD45, CD3, CD4 and CD8 T cells. HumanCD45+-gated, CD3+, CD4+ and CD8+ subsets were quantified with theTruCount tubes following the manufacturer's instructions.

In Vivo Study of Adoptive Cell Therapy

Female non-obese diabetic/severe combined immunodeficiency/γ-chain^(−/−)(NSG) mice 8 to 12 weeks of age were obtained from the Stem Cell andXenograft Core of the Abramson Cancer Center, University ofPennsylvania. The mice were inoculated subcutaneously with 3×10⁶ fLuc⁺SKOV3 cells on the flank on day 0. Four or Five mice were randomized pergroup before treatment. After tumors became palpable, human primary Tcells were activated and transduced as described previously. T cellswere expanded in the presence of IL-2 (5 ng/mL) for about 2 weeks. Whenthe tumor burden was ˜250-300 mm³, the mice were injected with 5×10⁶CAR-T cells or 100 μl saline intravenously and then received dailyintraperitoneal injection of 5 μg of IL-2, IL-7, IL-15, IL-18, IL-21 orphosphate buffer solution (PBS) for 7 days. Tumor dimensions weremeasured with calipers and tumor volumes were calculated with thefollowing formula: tumor volume=(length×width²)/2. The number andphenotype of transferred T cells in recipient mouse blood was determinedby flow cytometry after retro-orbital bleeding. The mice were euthanizedwhen the tumor volumes were more than 2000 mm³ and tumors were resectedimmediately for further analysis.

Statistical Analysis

Statistical analysis was performed with Prism 5 (GraphPad software) andIBM SPSS Statistics 20.0 software. The data were shown as mean±SEMunless clarified. Paired sample t-tests or nonparametric Wilcoxon ranktests were used for comparison of two groups and repeated measures ANOVAor Friedman test were used to test statistical significance ofdifferences among three or more groups. Findings were considered asstatistically significant when P-values were less than 0.05.

Results Construction and Expression of Anti-FRα C4 CAR

The pELNS-C4-27z CAR comprised of the anti-FRα C4 scFv linked to a CD8αhinge and transmembrane region, followed by a CD3ζ signaling moiety intandem with the CD27 intracellular signaling motif. Primary human Tcells were efficiently transduced with C4 CAR lentiviral vectors withtransduction efficiencies of 43%˜65% when detected at 48 h aftertransduction. The CAR expression levels were comparable between CD4+ andCD8+ T cells (52.6±10.2% vs. 49.5±17.1%, P=0.713).

Different Anti-Tumor Efficacy of Various Cytokines in Animal Models

This study examined whether the in vivo cytokine administration incombination with CAR-T cell injection could enhance the anti-tumoractivity of CAR-T cells. Mice bearing subcutaneous SKOV3 tumors receivedeither saline or 5×10⁶ C4-27z CAR-T cell intravenously injection on day39. Compared with saline group, mice receiving CAR-T cell therapyunderwent short-time tumor regression and the tumor began to reboundedfrom day 56. Of the various cytokine groups, mice receiving IL-15 andIL-21 injection presented best tumor suppression, followed by IL-2 andIL-7, whereas IL-18 and PBS treated mice had the heaviest tumor burden.The persistence of transferred T cells in the peripheral blood wasdetermined 15 days after adoptive transfer and when termination. Highestnumbers of CD4+ and CD8+ T cells were detected in mice treated withIL-15, followed by IL-21. The day+15 CD4+ and CD8+ T-cell count wereconsistent with the tumor regression and predicted the final tumorweight. The mice were killed 73 days after tumor challenge and thetumors were analyzed for the presence of human T cells. Similarly withperipheral blood, mice treated with IL-15 presented highest T cellnumber in the tumor, followed by IL-21, IL-2, IL-7, PBS and IL-18. Theratios of CD4 to CD8 were comparable among different cytokine groups,with a predomination of CD4+ T cells both in blood and tumor in allcytokine groups. The CAR expression in CD8+ T cells were comparableamong the above groups (45.1%˜62.4%), while IL-15 and IL-21 groups hadhigher proportions of CAR+ CD4 T cells than IL-2 and IL-18 groups. As tothe phenotype, all the CAR-T cells in the tumor were CD62L⁻ and CCR7⁻,while 35%˜60% of them expressed CD45RA+ (Temra) (data not shown). CD8+ Tcells were more likely to retained CD27 expression while the CD28expression was comparable between CD4+ and CD8+ T cells.

Example 4: Improvement in the Efficacy of Adoptively Transferred T Cellsby the Genetic Blockade of Protein Kinase A (PKA) Function

The protein kinase A (PKA) holoenzyme is a heterotetramer consisting of2 regulatory subunits (RI and RII) and 2 catalytic subunits (CI andCII). Upon activation by cAMP, the R subunits dissociate, and the Csubunits proceed to phosphorylate a large myriad of target substrates;the cAMP-PKA signaling cascade is one of the most ubiquitous andwell-established second messenger systems to date. Hence, it followsthat the attenuation of cAMP signaling in T cells may represent a meansto prolong TCR-mediated signaling and better killing capacity.

In order for PKA to elicit its functions, it is tethered to lipid raftsin close proximity to adenylyl cyclase, the enzyme that metabolizes ATPto generate cAMP. The tethering and subsequent compartmentalization ofPKA and its effects are mediated by A-kinase anchoring proteins (AKAP).AKAP therefore serve as a platform on which cAMP and PKA signalingconverge, and provide temporal and spatial regulation of these entities.With regard to T cell signaling PKA is directed to the TCR by binding toa protein called Ezrin, which inserts into the membrane and serves asthe AKAP.

The RIAD or RISR-RIAD peptide binds to the RI subunit of PKA with highaffinity and disrupts PKA anchoring to Ezrin, thereby neutralizing PKAsignaling (Carlson et al., 2006). Normally, cAMP binds to PKA, whichactivates it. The PKA binds to Ezrin and is brought in contact with thekinase Csk. Csk is activated which then phosphorylates and inactivatesLck which stops TCR signaling. With RIAD or RISR-RIAD around, thePKA-cAMP complex cannot localize to Ezrin and thus does not have accessto Csk. The RIAD-mediated displacement of PKA ultimately diminishesphosphorylation of Tyr-505 on Lck, and hence upregulates TCR signaling.

An additional peptide sequence enhances RIAD binding to PKA, therebyaugmenting the release of T cell inhibition by cAMP; this additionalpeptide is designated RISR (RI specifier region). A transgenic mousemodel expressed RISR-RIAD in T cells under the control of the lck distalpromoter displaced PKA from lipid rafts of T cells. These mice showedheightened TCR signaling and interleukin 2 (IL2) secretion, andresistance to PGE2. Impressively, these mice were also more resistant tomurine AIDS.

The approach described herein was to couple T cell anti-tumor activitywith RISR-RIAD. MesoCAR-expressing viral vectors with RISR-RIAD weregenerated to test the efficacy of these constructs against tumor cellsin vitro and in vivo (FIG. 1).

PKA regulates T cell signaling by phosphorylating the kinase Csk atS364, which activates this kinase, resulting in phosphorylation of thekey TCR proximal signaling molecule Lck at Y505, which inhibits itsactivity. FIG. 2 is a blot showing RISR-RIAD expression preventedphosphorylation of Csk-S364 and Lck-Y505. RISR-RIAD expression resultedin more active signaling at baseline and after TCR stimulation (FIG. 3).

Example 5: Retroviral Transduction of Activated Murine T Cells with themesoCAR-RISR-RIAD Construct LED to Better Killing of Tumor Cells InVitro and In Vivo Compared to MesoCAR T Cells

A technique to successfully transduce murine T cells with CARs usingmodified retroviruses was developed. In the case of themesoCAR-RISR-RIAD construct, the RISR-RIAD transgene was tagged by mycand flag, which allowed for simultaneous detection with mesoCAR.Compared to T cells transduced with mesoCAR,mesoCAR-RISR-RIAD-transduced T cells exhibited higher killing ability asdemonstrated by an overnight in vitro killing assay ofmesothelin-expressing AE17 murine mesothelioma cells (AE17meso) indiffering effector-to-target ratios (E:T) shown in FIG. 4. Bothconstructs were shown to be target-specific as little to no killing wasobserved in the ova-expressing cell line (AE17ova).

MesoCAR-expressing T cells exhibited modest killing ability and weresusceptible to suppression by adenosine and PGE2; two agents were shownto exert their inhibitory effects in a cAMP-dependent manner FIGS. 5Aand 5B substantiated that observation. FIGS. 5A and 5B further showedthat the killing ability of mesoCAR-RISR-RIAD construct was unaffectedby these inhibitory molecules, adenosine and PGE2.

Given the key role of PKA signaling in the inhibition of T cellfunction, it was hypothesized that cloning the RISR-RIAD transgene intoT cells expressing chimeric antigen receptors or transgenic T cellswould enhance their function within the tumor microenvironment, andresult in superior tumoricidal ability as compared to CAR T cells, ortransgenic TCR T cells alone. The hypothesis was tested in mouse modelsof mesothelioma and lung cancer treatment. Both murine and human CAR Tcells were used, and two different tumor antigens were targeted,mesothelin and the stromal antigen fibroblast activation protein (FAP),see FIG. 6. The effect of RISR-RIAD in human transgenic Ly95TCR-transduced T cells targeted to the tumor antigen NY-ESO1 expressedon HLA-A2-expressing lung cancer cells was also tested. RISR-RIADtransgene in T cells expressing chimeric antigen receptors or transgenicT cells enhanced their function within the tumor microenvironment, andresulted in superior tumoricidal ability as compared to CAR T cells, ortransgenic TCR T cells alone. This hypothesis was tested in mouse modelsof mesothelioma and lung cancer treatment.

Example 6: Retroviral Transduction of Activated Murine T Cells with themesoCAR-RISR-RIAD Construct Leads to Enhanced Killing of Tumor Cells InVitro Due to Dampened PKA-Mediated Signaling

The effect of the RISR-RIAD construct in CAR T cells that was generatedfrom murine lymphocytes was studied. Using modified retrovirusesexpressing mesoCAR, with and without the RISR-RIAD transgene, activatedmouse T cells were successfully transduced with over 50% transductionefficiency. The detection of the RISR-RIAD transgene was accomplishedusing antibodies to detect either the myc or ddk tag (FIG. 7A). Theireffector functions were assessed at differing effector-to-target (E:T)ratios of transduced T cells by co-culturing them overnight withova-expressing and mesothelin-expressing AE17 murine mesothelioma cells(AE17ova and AE17meso, respectively). Compared to murine T cellstransduced with mesoCAR alone, mesoCAR-RISR-RIAD T cells showedincreased killing ability (FIG. 7B) and IFNγ production with littleeffect on AE17ova cells (FIG. 7C). Additionally, mesoCAR-RIAD T cellswere markedly more effective in reducing tumor volume as compared tomesoCAR T cells (FIG. 23).

The resistance of mesoCAR-RISR-RIAD T cells to immunosuppressionmediated by adenosine and PGE2 was tested. The expected dose-dependentinhibition of the killing ability of mesoCAR T cells in the presence ofimmunosuppressive adenosine and PGE2 was observed, but notmesoCAR-RISR-RIAD T cells (FIG. 7D).

Example 7: Intravenous Administration of Murine mesoCAR-RISR-RIAD TCells Controls Tumor Progression More Efficiently than mesoCAR T Cells

Two million AE17meso cells were injected subcutaneously in the flanks ofwild-type C57Bl/6 mice, and after tumors were established (about 7-10days after inoculation), 10⁷ mesoCAR or mesoCAR-RISR-RIAD murine T cellswere administered intravenously. Tumor growth was monitored for the next10-14 days using caliper measurements. The tumor control was observedwith mesoCAR T cells; however, mesoCAR-RISR-RIAD T cells hadsignificantly reduced growth of AE17meso tumors compared to mesoCAR Tcells, p≤0.01 (FIG. 8A).

To evaluate the efficacy of RISR-RIAD in a completely different CARsystem, a construct that targets fibroblast activation protein (FAP)present on stromal cells surrounding the tumor was used. C57Bl/6wild-type mice were inoculated with the pancreatic cell line, PDA4662;this cell line induces a highly dense stromal environment.PDA4662-bearing mice were subsequently treated with 10⁷ FAPCAR orFAPCAR-RISR-RIAD T cells when the tumor burden was approximately 200mm³. Fourteen days post-adoptive transfer, FAPCAR-RISR-RIAD T cellsshowed significantly enhanced anti-tumor activity in this model (FIG.8B).

Example 8: MesoCAR-RISR-RIAD T Cells Show Increased Migration In Vitroand Enhanced Trafficking into Tumors In Vivo

In order to determine the mechanisms involved in the enhanced anti-tumoreffects of murine mesoCAR-RISR-RIAD T cells, flow cytometry analysis ofspleen- and tumor-infiltrating lymphocytes at Day 3 post-adoptivetransfer was performed. The analysis of murine CAR T cells freshlyisolated from AE17meso tumors in mice revealed that in comparison withmesoCAR-treated tumors, mesoCAR-RISR-RIAD-treated tumors showed anincreased influx of CD8 cells (FIG. 9A), while analysis of spleens fromtumor-bearing mice showed an increase in CD4 number in themesoCAR-RISR-RIAD treatment group (FIG. 9B). Since this observationrecapitulates what was observed in human mesoCAR-RISR-RIAD T cells(FIGS. 16A-16C), i.e., they appeared to migrate to tumor sites much moreefficiently than mesoCAR T cells, transmigration assays were performedusing 0.5 μm transwell inserts in which transduced T cells were allowedto traffic toward the chemokine IP10 in the lower well. Higherchemotaxis and IP10-mediated migration rate was consistently observed inmesoCAR-RISR-RIAD T cells compared to mesoCAR T cells (FIG. 9C), whichlikely contributes to more robust tumor control. This increasedefficiency in migration is attributed to integrin 131-mediated signalingas denoted by the high expression of CD29 in mesoCAR-RISR-RIAD T cells(FIG. 9D). No changes in CXCR3 or CD11A expression was observed.

Example 9: Lentiviral Transduction of Activated Human T Cells with themesoCAR-RISR-RIAD Construct LED to Better Killing of Tumor Cells InVitro and In Vivo Compared to MesoCAR T Cells

In a similar manner, human T cells with lentiviruses expressing mesoCARand mesoCAR-RISR-RIAD were transduced to compare their killingefficiencies both alone and in the presence of immunosuppressive agentssuch as adenosine. Both T cells were incubated withmesothelin-transduced human mesothelioma cells (EM-meso) at varying E:T.FIG. 10 shows that at a E:T of 10:1, mesoCAR-RISR-RIAD T cells have moreactivity at baseline and possess far superior killing ability in vitroin spite of the presence of adenosine.

Example 10: Lentiviral Transduction of Activated Human T Cells with themesoCAR-RISR-RIAD Construct Leads to Enhanced Killing of Tumor Cells InVitro

The development and use of CAR constructs expressing the scFv fromanti-human mesothelin, along with the human CD3ζ and 4-1BB cytoplasmicdomains (mesoCAR) has previously been reported; T cells transduced withthis construct cured some mesothelioma cell lines in mice, but notothers due to T cell hypofunction exerted by an immunosuppressive solidtumor microenvironment.

In an attempt to overcome this issue, one of the inhibitory elements wasdisarmed, namely the cAMP-PKA pathway. Using modified lentivirusesexpressing mesoCAR, with and without the RISR-RIAD transgene, activatedhuman T cells were successfully transduced with over 30% transductionefficiency; the detection of the RISR-RIAD transgene was accomplishedusing antibodies to detect either the myc or ddk tag (FIG. 11A). Inorder to assess the effector functions of these T cells, differingeffector-to-target (E:T) ratios of transduced T cells were co-culturedovernight with human mesothelioma cells expressing mesothelin andluciferase (EMmeso-luc). Compared to T cells transduced with mesoCARalone, mesoCAR-RISR-RIAD T cells showed enhanced killing ability (FIG.11B) and higher interferon-γ (IFNγ) production in a dose-dependentmanner (FIG. 11C); both constructs are target-specific as little to nokilling was observed with EM parental (EMP) cells not expressingmesothelin.

Example 11: Primary Human T Cells Transduced with mesoCAR-RISR-RIAD areMore Resistant to Immunosuppression and Hypofunction Compared to mesoCART Cells

The resistance of mesoCAR-RISR-RIAD T cells to immunosuppressionmediated by adenosine and PGE2 was tested. An overnight co-culture assay(at differing E:T ratios) was set up in the presence/absence of varyingdoses of adenosine and PGE2. As expected, a dose-dependent inhibition ofthe killing ability of mesoCAR T cells was observed in the presence ofadenosine and PGE2, but mesoCAR-RISR-RIAD T cells were virtuallyunaffected by these inhibitory molecules (FIG. 12 shown at 5:1 E:T), aswell as having enhanced activity in the absence of these inhibitors(FIG. 12).

Example 12: TCR Signaling is Enhanced at Baseline and after Stimulationin mesoCAR-RISR-RIAD T Cells

To evaluate signaling, T cells were examined at baseline and after 20minutes of exposure to plate-bound CD3 and CD28 antibodies. At baseline,compared with mesoCAR T cells, mesoCAR-RISR-RIAD T cells showed someevidence of activation with increased phosphorylation of Erk,Lck^(Y394), and Akt. After stimulation, the expected increases in Erk,Lck^(Y394), and Akt phosphorylation in mesoCAR T cells were observed,but in mesoCAR-RISR-RIAD T cells, higher levels of phosphorylation wereobserved (FIG. 13A).

Example 13: PKA Signaling is Attenuated in mesoCAR-RISR-RIAD T Cells

As described herein, one way in which PKA regulates T cell signaling isby phosphorylating the kinase Csk at S364, which activates it andresults in phosphorylation of the key TCR proximal signaling moleculeLck at Y505, which inhibits its activity. To confirm that this mechanismof RISR-RIAD inactivation was operative in the cells, thephosphorylation status of Csk^(S364), and Lck^(Y505) was assessed. Theloss of phosphorylation was observed at both these residues at baselineand after CD3/28 stimulation in mesoCAR-RISR-RIAD T cells compared tomesoCAR T cells (FIG. 13B).

Example 14: Human T Cells with the mesoCAR-RISR-RIAD Construct haveEnhanced Ability to Kill Tumors In Vivo

To compare the ability of human mesoCAR-RISR-RIAD T cells in controllingtumor burden to that of mesoCAR T cells, EMmeso cells weresubcutaneously inoculated on the flanks of immunodeficient NSG mice, andwhen tumors were around 200 mm³ in volume, 10⁷ mesoCAR- ormesoCAR-RISR-RIAD-expressing primary human T cells were intravenouslyadministered. EMmeso-bearing NSG mice treated with mesoCAR T cellsshowed significantly slower tumor progression by approximately 40%(p≤0.001) compared to untreated tumors; however, injection ofmesoCAR-RISR-RIAD T cells significantly enhanced the mesoCAR anti-tumoreffect (p≤0.0001), resulting in tumors that were 60% smaller compared tountreated tumors (FIG. 14A).

Example 15: Human mesoCAR-RISR-RIAD Tumor-Infiltrating T Cells ShowIncreased Persistence and Increased Activity

To better understand the mechanisms of the enhanced anti-tumor effectsobserved with mesoCAR-RISR-RIAD T cells, EMmeso-bearing mice weresacrificed on Day 32 after T cell administration, and their tumors werepooled and processed as described herein. Flow cytometry analysis oftumors at this time point showed increased numbers of CD8 cells withinthe tumors of mesoCAR-RISR-RIAD-treated mice compared to mesoCAR-treatedmice (FIG. 14B). Adoptively transferred human T cells were isolated fromthese tumors and reacted them with freshly plated tumor cells inculture. This was done to determine the ex vivo ability of these T cells(“at harvest”) to kill tumor cells and secrete IFNγ as compared with theT cells that were originally administered at Day 0 (“cryo”). As shown inFIG. 14C (Cryo group in both graphs), mesoCAR-RISR-RIAD T cells hadhigher baseline killing activity and secreted more IFNγ than mesoCAR Tcells. MesoCAR tumor-infiltrating lymphocytes (TILs) developedhypofunction with respect to both killing and IFNγ secretion (FIG. 14C,At harvest in both graphs); in marked contrast, mesoCAR-RISR-RIAD TILsretained almost full cytolytic and IFNγ production capacity. Again,after mesoCAR TILs were allowed to rest in culture medium for 24 hours(“after overnight rest”), they recovered almost full activity whilemesoCAR-RISR-RIAD T cells remained active (FIG. 14C, After overnightrest in both graphs). ELISA showed that mesoCAR-RIAD T cells, eitherfreshly isolated from these tumor-bearing mice, or after overnightculture in medium, produced more interferon-γ compared to mesoCAR Tcells alone (FIG. 23).

Example 16: In Vitro Killing of Tumor by Ly95-RISR-RIAD T Cells

To evaluate the efficacy of RISR-RIAD in a CAR-free system, theNY-ESO1-reactive Ly95 TCR construct, an affinity-enhanced variant of thewild-type IG4 TCR, was expressed in human T cells with and withoutRISR-RIAD. The Ly95-RISR-RIAD T cells demonstrated enhanced tumorkilling at each E:T ratio (FIG. 15). The Ly95-RISR-RIAD T cells werealso less susceptible to inhibition by low (FIG. 16) and high dose (FIG.17) PGE2 and adenosine (FIG. 18).

Ly95-RISR-RIAD T cells also produced more IFNgamma than Ly95 T cellsunder adenosine (FIG. 19), low dose (FIG. 20), and high dose (FIG. 21)PGE2 suppression conditions.

Example 17: Use of a Modified RIAD/RISR Based on the Endogenous EzrinSequence

Depending on the HLA type of the donor, the RIAD-RISR protein couldcontain epitopes that might be presented on the surface of the T cellsand induce CD4 or CD8 host responses resulting in a potential loss ofthe T cells by immune attack. In silico analysis of the RISR/RIADconstruct of this invention showed no immunogenic neoepitopes for commonHLA types. To preclude any chance of immune reaction, another form ofthe RIAD-RISR construct was designed based upon the sequence of theendogenous protein Ezrin. The sequence of this alternate form of theRIAD-RISR is highly similar to the above described RISR-RIAD protein(SEQ ID NOs: 63-65) but has lower chance, if any, to be immunogenic asit comprises an identical sequence to the endogenous Ezrin sequence.

The sequence of the RISR-RIAD alternate construct is described hereinbelow.

In one aspect, the fundamental part of the construct is an Ezrinsequence (Black bolded, highlighted in light and dark grey, in a box),having an amino acid sequence ofQMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEADRMAALRAKEELERQAVDQIKSQEQLAAELAEYTAKIALL (SEQ ID NO: 114) and a nucleicacid sequence of CAGATGATGCGCGAAAAAGAAGAACTGATGCTGCGCCTGCAGGATTATGAAGAAAAAACCAAAAAAGCGGAACGCGAACTGAGCGAACAGATTCAGCGCGCGCTGCAGCTGGAAGAAGAACGCAAACGCGCGCAGGAAGAAGCGGAACGCCTGGAAGCGGATCGCATGGCGGCGCTGCGCGCGAAAGAAGAACTGGAACGCCAGGCGGTGGATCAGATTAAAAGCCAGGAACAGCTGGCGGCGGAACTGGCGGAATATACCGC GAAAATTGCGCTGCTG(SEQ ID NO: 115). In some embodiments, the construct comprises somespecific enzyme restriction sites such as Xba1 (Grey color) and BSpel(Grey color and underlined). In other embodiments, the constructcomprises other suitable enzyme restriction sites known in the art. Insome embodiments, tag sequences are inserted in order to permit trackingthe alternate RISR-RIAD protein. Non limiting examples of tag sequencesfor tracking a protein are Myc Tag (Black color and inclined), “Flag”(DDK) tag (Black color and underlined) and Human influenza hemagglutinin(HA) tag.

In another aspect, similar to the RIAD-RISR construct describedpreviously herein (SEQ ID NOs: 63-65), the nucleic acid sequences ofthis alternate RIAD-RISR construct (SEQ ID NO: 116) are cloned into aCAR or TCR expressing vector and augment efficacy of adoptivetransferred T cells therapy as described previously herein.

The detailed nucleic acid and amino acid sequences for the RISR-RIADalternate construct is listed below herein:

(SEQ ID NO: 117)

(SEQ ID NO: 118)

Nucleic acid sequence of the entire RISR,  SEQ ID NO: 119CAGATGATGCGCGAAAAAGAAGAACTGATGCTGCGCCTGCAGGATTATGAAGAAAAAACCAAAAAAGCGGAACGCGAACTGAGCGAACAGATTCAGCGCGCGCTGCAGCTGGAAGAAGAACGCAAACGCGCGCAGGAAGAAGCGGAACGCCTGGAAGCGGATCGCATGGCGGCGCTGCGCGCGAAAGAAGAACTGGAACGCCAGGCGGTG GATCAGATTAmino acid sequence of the entire RISR,  SEQ ID NO: 120QMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEADRMAALRAKEELERQAVDQI

Nucleic acid sequence of RIAD,  SEQ ID NOs: 121TAAAAGCCAGGAACAGCTGGCGGCGGAACTGGCGGAATATACCGCGAAAATTG CGCTGCTGAmino acid sequence of RIAD,  SEQ ID NO: 122 KSQEQLAAELAEYTAKIALLNucleic acid sequence of RISR-RIAD (Ezrin),  SEQ ID NO: 123CAGATGATGCGCGAAAAAGAAGAACTGATGCTGCGCCTGCAGGATTATGAAGAAAAAACCAAAAAAGCGGAACGCGAACTGAGCGAACAGATTCAGCGCGCGCTGCAGCTGGAAGAAGAACGCAAACGCGCGCAGGAAGAAGCGGAACGCCTGGAAGCGGATCGCATGGCGGCGCTGCGCGCGAAAGAAGAACTGGAACGCCAGGCGGTGGATCAGATTAAAAGCCAGGAACAGCTGGCGGCGGAACTGGCGGAATATACCGC GAAAATTGCGCTGCTGAmino acid sequence of RISR-RIAD (Ezrin),  SEQ ID NO: 124QMMREKEELMLRLQDYEEKTKKAERELSEQIQRALQLEEERKRAQEEAERLEADRMAALRAKEELERQAVDQIKSQEQLAAELAEYTAKIALL

Example 18

Adoptive T cell therapy utilizing chimeric antigen receptors (CARs) hasbeen used to treat hematologic malignancies and solid tumors. Loss of Tcell effector function occurs in endogenous T cells, and the samephenomenon with intravenously-administered human CAR T cells has beenobserved. The data described herein highlight the importance ofbolstering adoptively transferred T cell resistance to immunosuppressionencountered within the tumor microenvironment.

To address immunosuppression mediated by adenosine and PGE2, twoimportant well-established inhibitory factors within the tumormicroenvironment that dampen the anti-tumor response, were analyzedherein. Like many other molecules in the tumor microenvironment, boththese entities facilitate signal transduction through their cognateG-protein coupled receptors that culminate in the accumulation of cAMP,a potent mediator of immunosuppression via the effector protein PKA.

The discovery and development of RISR and RIAD brought forth thehypothesis that the co-expression of the RISR-RIAD transgene with a CAR-and transgenic TCR-transduced T cells may protect them fromcAMP-mediated immunosuppression. Therefore in this study, the anti-tumorefficacy of CAR and transgenic TCR T cells co-transduced with thetransgene RISR-RIAD in murine and human TILs was investigated.

Using activated human T cells that were transduced withmesoCAR-RISR-RIAD using modified lentiviruses, it was demonstrated invitro that human mesoCAR-RISR-RIAD T cells exhibited antigen-specificcytotoxicity against EMmeso cells, leaving the parental EM cell linerelatively unaffected (FIG. 11B). Moreover, the extent of EMmesocytolysis by mesoCAR-RISR-RIAD T cells was greater than that of mesoCART cells alone (FIG. 11B); this corresponded to the heightened generationof IFNγ by mesoCAR-RISR-RIAD T cells (FIG. 11C). In order to evaluatethe contribution of the RISR-RIAD transgene to the system, the in vitrocytolysis assay was challenged with the addition of adenosine and PGE2,and observed the superior resistance of mesoCAR-RISR-RIAD T cells tothese immunosuppressive entities in comparison to the dose-dependentinhibition of mesoCAR killing of EMmeso cells (FIG. 12). Since RISR-RIADwas reported to specifically disrupt PKA anchoring to the membrane, andhence abrogate inhibitory PKA signaling,

The effect of RISR-RIAD on the two key TCR-related, PKA-affectedproximal signaling molecules, Csk and Lck, was assessed. The loss ofinhibitory PKA signaling in mesoCAR-RISR-RIAD T cells—the loss ofphosphorylation of Csk at S364—was observed, and hence its inability tophosphorylate (and activate) Lck at Y505 confer resistance of humanmesoCAR-RISR-RIAD T cells to inhibitory cAMP signaling (FIG. 13B).

The status of actual T cell signaling components (pLck^(Y394), pERK, andpAkt) after engagement of the TCR by CD3/CD28-mediated activation wasassessed. As expected, increased signaling after TCR stimulation wasobserved. However, somewhat surprisingly, even at baseline,mesoCAR-RISR-RIAD T cells showed greater activity compared to mesoCAR Tcells, as evidenced by increased pERK, pLck^(Y394), and pAktphosphorylation. This suggests that there is tonic inhibitory PKAactivity in the system, even in resting effector T cells. Overall, thesedata suggest that mesoCAR-RISR-RIAD T cells would show improved functionin an immunosuppressive microenvironment.

To test this theory, immunodeficient NSG mice bearing established EMmesotumors with both mesoCAR and mesoCAR T cells were treated, and superiortumor volume reduction upon treatment with mesoCAR-RISR-RIAD T cells wasobserved in comparison with mesoCAR T cells (FIG. 14A). To delineatethis effect, TILs were evaluated at Day 32 after T cell administration,and saw a large increase in mesoCAR-RISR-RIAD T cell number within thetumor when compared to mesoCAR T cells (FIG. 14B). The lack of mesoCAR Tcell efficacy ex vivo was due to hypofunction acquired within the tumormicroenvironment as these T cells encounter a myriad of inhibitoryfactors; this hypofunction phenomenon is reversible as upon overnightrecovery (and removal of inhibitory factors) in complete cell culturemedium, mesoCAR T cells regain their cytolytic ability againstantigen-expressing tumor cells (FIG. 14C). However, mesoCAR-RISR-RIAD Tcells, in accordance with RISR-RIAD-conferred protection againstimmunosuppression, continued to kill EMmeso cells and generate highlevels of IFNγ even at time of harvest (FIG. 14C).

The application of the RISR-RIAD transgene in murine mesoCAR T cells wasextended both in vitro and in vivo, and observed similar effects as inthe human system—enhanced cytolysis, along with increased murine IFNγgeneration, and resistance to adenosine- and PGE2-facilitatedsuppression in cytolysis (FIGS. 7A-7D). In vivo, the expression of theRISR-RIAD transgene in both mesoCAR- and FAPCAR-transduced murine Tcells successfully slowed tumor growth to a greater extent as comparedto non-RISR-RIAD-expressing CAR T cells (FIGS. 8A-8B). Flow cytometryanalysis of murine TILs similarly denoted an increased influx ofmesoCAR-RISR-RIAD T cells within the spleen and tumor (FIGS. 9A and 9B)as previously seen in the human system (FIGS. 7A-7D). Given theseobservations, the possibility that mesoCAR-RISR-RIAD cells may possesssuperior migration ability over mesoCAR cells, and hence, can betterinfiltrate and kill tumors was investigated. Transmigration assays usingthe chemokine IP10 revealed better mesoCAR-RISR-RIAD trafficking in aCD29 (integrin β1)-dependent fashion (FIGS. 9C and 9D).

Taken together, the study showed that CAR-RISR-RIAD T cells werespecifically activated, killed target antigen-expressing tumor cells invitro and in vivo with no obvious toxicity, and were far superior intheir cytolysis ability compared to CAR T cells alone. This additionalboon was due to the RISR-RIAD-conferred protection against cAMP-mediatedimmunosuppression within the tumor microenvironment, and can thereforebe used to inhibit tumor growth as a monotherapy, and may possessadditive or synergistic anti-tumor effects when combined with othertumor cell-directed therapies; adoptive T cell use in the treatment ofsolid tumors must be resistant to the immunosuppressive milieu. Theinsertion of the RISR-RIAD transgene can be easily accomplished into anyCAR or T cells with transgenic TCRs in a bicistronic fashion.

The materials and methods employed in these experiments are nowdescribed.

Generation of RISR-RIAD-Expressing mesoCAR, FAPCAR, and Transgenic Ly95TCR Constructs, and T Cell Generation

The RISR-RIAD construct, incorporated with myc and ddk (FLAG) tags, wassynthesized by Integrated DNA Technologies in the pIDT.SMART cloningplasmid. It was then subcloned into a human mesoCAR-expressing MigR1retroviral vector, dubbed MigR1.mesoCAR-RISR-RIAD (FIG. 6), and murineFAPCAR-expressing MigR1 retroviral vector, dubbed MigR1.FAPCAR-RISR-RIAD(FIG. 6). Primary murine T cells were isolated and transduced with theseretroviral particles as previously described (Riese, et al., Canc. Cell,2013, 3566-3577). Similarly, RISR-RIAD and mesoCAR cDNA were subclonedinto the lentiviral vector pTRPE (dubbed pTRPE.mesoCAR andpTRPE.mesoCAR-RISR-RIAD, shown in FIG. 1C). The isolation, beadactivation, transduction using pTRPE.mesoCAR andpTRPE.mesoCAR-RISR-RIAD, and subsequent expansion of primary human Tcells were carried out as previously described (Moon, et al., Clin.Canc. Res., 2014, 20:4262-4273). The RISR-RIAD transgene was alsoinserted into the NY-ESO1-reactive Ly95 transgenic TCR construct.

Generation of the Target Lung Cancer Cell Line

The human lung cancer cell line A549-CBG was generated by stablytransducing the A549 cell line (ATCC CCL185) with a lentiviral vectorencoding Click Beetle Green (CBG) and GFP (CBG-T2A-GFP) and flow-sortedto 100% GFP positivity. The sorted A549-CBG cell line was thentransduced by a retroviral vector encoding NY-ESO-1-T2A-HLA-A2. Thetransduced A549-CBG cells were subjected to limiting dilution at 0.5cell per well in 96-well plates. Resulting clones were tested by flowcytometry for HLA-A2 expression. HLA-A2 positive clones were selectedand tested by co-culture with T cells expressing the NY-ESO-1 TCR. Theclones expressing HLA-A2 that could stimulate NY-ESO-1 TCR-expressing Tcells to secrete IFNγ were pooled to generate the A549-NY-ESO-1-A2-CBG(A549-A2-ESO) cell line.

Animals

Studies using retroviral MigR1-transduced T cells were carried out inwild-type C57Bl/6 mice obtained from Charles River Laboratories. Studiesusing lentiviral pTRPE-transduced human T cells were conducted inNOD/scid/IL2rγ^(−/−) (NSG) mice bred at the Children's Hospital ofPhiladelphia. All test animals used were females at 10-12 weeks of age.

Cell Lines

All cell lines used were cultured as previously described (Moon, et al.,Clin. Canc. Res., 2014, 20:4262-4273), and routinely examined forMycoplasma infection using the MycoAlert kit (Lonza #LT07-318). AE17murine mesothelioma cells expressing chicken ovalbumin (dubbed AE17ova)were obtained from the University of Western Australia, while EM humanmesothelioma cells were derived from a patient's tumor (dubbedEMparental, or EMP). These cell lines, stably transduced with mesothelin(AE17meso and EMmeso, respectively), were used for in vivo studies withmesoCAR T cells. Murine 3T3Balb/c cells (3T3parental, or 3T3P) werepurchased from American Type Culture Collection, and stably transducedwith FAP (Riese, et al., Canc. Cell, 2013, 3566-3577); these were usedfor studies with FAPCAR T cells. In vitro studies were also performedusing these cell lines that were additionally transduced to expressfirefly luciferase, named AE17ova-luc, AE17meso-luc, EMP-luc,EMmeso-luc, 3T3P-luc, and 3T3FAP-luc. The murine 4662 pancreatic ductalcarcinoma cell line (PDA4662) were derived from an autochthonouspancreatic tumor isolated from a fully backcrossed C57BL/6Kras^(G12D):Trp53^(R172H):Pdx-1 Cre (KPC) mouse.

Antibodies

The detection of RISR-RIAD was carried out using anti-myc antibodies(Cell Signaling Technologies #3739 for flow cytometry, and #2722 forwestern blotting) and anti-ddk antibodies (Biolegend #637307 for flowcytometry and Origene #TA50011 for western blotting). The followingconjugated antibodies for flow cytometric detection of murine cells werepurchased from Biolegend: CD206 (#141720), CD8 (#100762), CD4 (#100406),IFNγ (#505825), IL2 (#503808), and anti-GFP (#338008); BD Biosciences:Ly6G (#551480), CD19 (#561739), B220 (#553091), CD69 (#552879), and CD44(#553135); and eBiosciences: F4/80 (#17-4801-82), CD11B (#12-0112-82),CD3 (#48-0031-82), FOXP3 (#12-5773-80), and 4-1BB (#17-1371-80). For thedetection of human cells, the following conjugated antibodies for flowcytometry were purchased from Biolegend: FOXP3 (#320106); BDBiosciences: CD25 (#555432), CD45 (#555483), CD8 (#555367), IL2(#340448), CD69 (#555530), and TNFα (#340511); and R&D Biosystems: humanmesothelin (FAB32652P).

Assessment of T Cell Effector Functions

Functional assays performed to characterize persistence and activity ofendogenous and genetically modified T cells in vitro, in vivo, and exvivo are outlined below. All assays are performed at least thrice in anindependent fashion, unless otherwise noted.

In vitro cytotoxicity and interferon-γ (IFNγ) ELISA

-   -   Triplicates of luciferase-expressing parental and        antigen-expressing cell lines were co-cultured with differing        ratios of CAR-expressing T cells to tumor cells as previously        described in 96-well plates overnight; cytotoxicity of T cells        were evaluated the following day, and culture supernatants were        collected for IFNγ ELISA. In order to evaluate the resistance of        RISR-RIAD-expressing T cells to immunosuppression, in vitro        cytotoxicity assays were also performed in the presence of PGE2        (Enzo Life Sciences #BML-PG007), and adenosine (Sigma #A9251).

In vivo studies

-   -   For mesoCAR and FAPCAR studies in wild-type C57Bl/6 mice, 2        million AE17meso and PDA4662 cells were subcutaneously        inoculated as previously described (Riese, et al., Canc. Cell,        2013, 3566-3577: Wang, et al., Canc. Cell Immunol. Res., 2014,        2:154-166). Similarly, for mesoCAR and Ly95 transgenic TCR T        cell studies in immunodeficient NSG mice, 2 million EMmeso and        A549-A2-ESO cells were subcutaneously inoculated. When tumors        were approximately 200 mm³, mice were given 10⁷ CAR-expressing,        or Ly95-expressing T cells via intravenous administration. Tumor        volume was monitored by caliper measurement of tumor diameter        twice weekly, and at different time points, tumor-bearing mice        were sacrificed for mechanistic studies using immunoblotting        and/or flow cytometry.

Immunoblotting

-   -   The phosphorylation status of various proximal T cell signaling        entities were assessed by western blotting (pERK, pAkt, pLck)        using a 1:3 ratio of CAR-expressing T cells and plate-bound        CD3/CD28 antibodies as previously described (Riese, et al.,        Canc. Cell, 2013, 3566-3577). Additionally, antibodies for the        detection of CD3z were purchased from Santa Cruz Biotechnologies        (#sc-1239), pLck^(Y505) from Cell Signaling Technologies        (#2751S), and pCsk^(S364) from Abcam (#ab61782).

Ex vivo T cell analysis

-   -   Tumors were harvested from mice, micro-dissected, and digested        in a mixture containing collagenase I, II, and IV, DNAse I, and        elastase in Leibovitz-L15 medium (Sigma #L1518) for 2 hours at        37° C. in a shaker incubator with 15-minute vortexing intervals        before filtering through 70-μm nylon mesh cell strainers.        Following this, procedures for red blood cell lysis, and tumor        single cell suspension isolation, along with spleen processing,        were carried out.

Flow cytometry (FACS)

-   -   Single cell suspensions were stained for surface and        intracellular markers using the previously listed antibodies        based on the manufacturers' recommendations. For intracellular        cytokine staining, cells were stimulated for 4-6 hours at 37° C.        in the presence of 0.7 μg/ml GolgiStop (BD Biosciences #554724)        with plate-bound 1 μg/ml anti-CD3 and 2 μg/ml anti-CD28        antibodies, and 30 ng/ml PMA and 1 μM ionomycin. Acquisition was        performed on a CyAn-ADP Analyzer (Beckman Coulter) or a BD        LSRFortessa (BD Biosciences). Data was analyzed using FlowJo        (TreeStar).

Transwell migration studies

-   -   Equal number of mesoCAR-GFP- and        mesoCAR-mCherry-RISR-RIAD-expressing cells were placed in 0.5 μm        polycarbonate transwell membranes, and allowed to migrate toward        PBS, cell culture medium alone, or with different concentrations        of the chemokine IP10, or tumor cell supernatant in tissue        culture plates. After 4 hours, remaining cells in the transwell        inserts and migrated cells in the bottom wells were collected,        counted, and stained for adhesion and migration markers for        analysis by flow cytometry.

Cell Culture Conditions

Tumor cells and T cells were cultured in RPMI 1640 (Gibco 11875-085)supplemented with 10% heat inactivated fetal calf serum (FCS), 100 U/mlpenicillin, 100 ug/ml streptomycin sulfate, and 1% L-glutamine (completecell culture medium).

Lentivirus Preparation

The NY-ESO1-reactive Ly95 TCR construct is an affinity-enhanced variantof the wild-type IG4 TCR identified from T cells recognizing the HLA-A2restricted NY-ESO-1:157-165 peptide antigen. In the mutant form, thethreonine at residue position 95 is substituted by leucine and theserine at residue position 96 is substituted by tyrosine. It wasconstructed using an overlapping PCR method based on the description andsequences published previously and incorporated into the lentiviralexpression vector pELNS bearing the EF1α promoter. Packaging of eachplasmid into lentivirus has been previously described. Titering oflentiviral concentration was performed by transduction of Sup-T1 cells(ATCC CRL-1942) at different virus dilutions and measurement oftransgenic TCR expression by flow cytometric analysis using ananti-human Vβ13.1 TCR chain antibody (Beckman Coulter, CA).

Isolation, Bead Activation, Transduction, and Expansion of Primary HumanT Lymphocytes

Primary human CD4⁺ T and CD8⁺ T cells were isolated from healthyvolunteer donors following leukopheresis by negative selection usingRosetteSep kits (Stem Cell Technologies, Vancouver, Canada). CD4⁺ andCD8⁺ T cells were mixed at a 1:1 ratio, and cultured in complete cellculture medium. They were stimulated with magnetic beads coated withanti-CD3/anti-CD28 at a 1:3 cell to bead ratio without the addition ofexogenous IL-2. T cells were transduced with lentiviral vectors at anMOI of approximately 5. Cells were counted and fed with complete cellculture medium every 2 days. Once they appeared to become quiescent asdetermined by both decreased growth kinetics and cell size, they wereused either for functional assays or cryopreserved. A small portion ofexpanded cells were stained for flow cytometry confirmation ofsuccessful Ly95 transduction using the Vβ13.1 TCR chain antibody.

Generation of the Target Lung Cancer Cell Line

The human lung cancer cell line A549-CBG was generated by stablytransducing the A549 cell line (ATCC CCL185) with a lentiviral vectorencoding Click Beetle Green (CBG) and GFP (CBG-T2A-GFP) and flow-sortedto 100% GFP positivity. The sorted A549-CBG cell line was thentransduced by a retroviral vector encoding NY-ESO-1-T2A-HLA-A2. Thetransduced A549-CBG cells were subjected to limiting dilution at 0.5cell per well in 96-well plates. Resulting clones were tested by flowcytometry for HLA-A2 expression. HLA-A2 positive clones were selectedand tested by co-culture with T cells expressing the NY-ESO-1 TCR. Theclones expressing HLA-A2 that could stimulate NY-ESO-1 TCR-expressing Tcells to secrete IFNγ were pooled to generate the A549-NY-ESO-1-A2-CBG(A549-A2-ESO) cell line.

FACS Analysis

Analysis of target tumor cells was conducted using APC-conjugatedantibody against HLA-A2 (BD Biosciences, CA), and a primary monoclonalantibody against NY-ESO-1 (Life Technologies, NY), followed by aPE-conjugated goat anti-mouse secondary antibody (BD Biosciences, CA).Analysis of expression of T cell surface markers was conducted usingfluorochrome-conjugated antibodies against CD45, CD8, CD4, PD-1 (BDBiosciences, CA), Tim-3 (eBioscience, CA), and Lag-3 (R&D systems, MN).Cells were stained in standard 5 ml round-bottom Falcon FACS tubes (BDBiosciences, CA) and analyzed on a 9-channel CyAn™ ADP Analyzer (BeckmanCoulter, CA).

Flow Cytometric T Cell Activation Assay

Analysis of T cell activation upon stimulation by target tumor cells wasperformed by plating Ly95 T cells with A549-A2-ESO or control A549-A2cells at 1:2 E:T ratio in round-bottom 96-well plates.Fluorochrome-conjugated antibodies against CD107a, granzyme B, and CD25(BD Biosciences, CA) were added to wells and incubated for 1 hour at 37°C. and 5% CO2 in the dark prior to the addition of Golgi Stop™ (BDBiosciences, CA). Measurement of IFNγ was performed using standardintracellular cytokine staining protocols.

In Vitro Testing of Tumor Cell Killing by Ly95 TCR T Cells

Control tumor cells and A549-A2-ESO cells were plated in a flat-bottom96-well plate at 5000 cells per well in triplicates. After overnightincubation at 37° C. and 5% CO2, Ly95 T cells were co-cultured atdifferent effector:target (E:T) ratios. After 18 hrs of incubation at37° C. and 5% CO2, supernatant from the wells were aspirated forcytokine analysis by ELISA, wells were washed, the remaining tumor cellswere lysed, and luminescence was read in a Modulus II MicroplateMultimode Plate Reader after addition of 100 ul of luciferin reagent(Promega E1501, Madison, Wis.). The same assay was used to examine thetumor killing ability of tumor-infiltrating lymphocytes obtained fromthe in vivo experiments.

Measurement of Ly95 T Cell IFNγ Secretion by ELISA

Supernatants from 18 hr tumor killing co-culture assays were prepared atdifferent dilutions and measured for levels of IFNγ by standard ELISAprotocol. (Biolegend, CA).

Animals

NOD/scid/IL2rγ^(−/−) (NSG) mice were bred in the Animal Services Unit ofthe Wistar Institute and the Children's Hospital of Philadelphia. Femalemice were used for experiments at 10 to 16 weeks of age.

In Vivo Xenograft Experiments

A total of 5×10⁶ A549-A2-ESO tumor cells were injected in the flanks ofNSG mice in a solution of X-Vivo media (Lonza, NJ) and Matrigel (BDBiosciences, CA). After tumors were established (100-200 mm³), the micewere randomly assigned to one of three intravenous (tail-vein) treatmentgroups: (i) saline, ii) 10×10⁶ non-transduced but expanded (NTD) Tcells, and iii) 10×10⁶ Ly95 T cells. In the experiments combininganti-PD-1 antibody with T cells, two additional groups were included:(iv) every 5-day intraperitoneal (IP) injection of 10 mg/kg anti-PD1antibody (Ultra-LEAF™, Biolegend, CA), and (v) 10×10⁶ Ly95 T cells IVplus every 5-day IP injection of 10 mg/kg anti-PD1 antibody. Tumors weremeasured using calipers and tumor volumes were calculated using theformula (π/6) (length)×(width)². When predefined protocol endpoints werereached, tumors were harvested, micro-dissected, and digested in asolution of 1:2 DNase:collagenase in a shaker incubator at 37° C. for 2hours. Digested tumors were then filtered through 70-μm nylon mesh cellstrainers, and red blood cells were lysed if needed (BD Pharm Lyse; BDBiosciences, CA). Spleens harvested from the same mice were alsofiltered through 70-μm nylon mesh cells trainers with red blood celllysis. 1×10⁶ cells from single-cell suspensions were placed in standardFACS tubes and were stained with anti-human CD45, CD8, CD4, andTCRVβ13.1 antibodies to assess degree of infiltration of adoptivelytransferred T cells. Additionally, cells were also stained withanti-PD1, anti-TIM3, and anti-LAGS antibodies to measure expression ofIRs on TILs. The in vivo experiments were repeated three times inindependent fashion. Groups contained 5-10 mice each.

Ex Vivo TIL Analysis

After digestion of harvested tumors, necrotic debris was first removedby processing the single cell suspension using a Dead Cell Removal Kit(Miltenyi Biotech, CA). TILs were subsequently isolated using ananti-human CD45-PE antibody (BD Biosciences, CA) with the EasySEP PESelection Kit (STEMCELL Technologies, Vancouver, Canada). Once isolated,functional analyses for TILs were performed in two different ways: (i)luciferase-based killing assays, and (ii) measurement of antigen-inducedT cell IFNγ secretion by ELISA (see above).

Animals

All animal experiment protocols were approved and conducted inaccordance with the Institutional Animal Care and Use Committee.NOD/scid/IL2rγ^(−/−) (NSG) mice were bred in the Animal Services Unit ofthe Wistar Institute and the Children's Hospital of Philadelphia. Femalemice were used for experiments at 10 to 16 weeks of age.

Statistical Analysis

All results were reported as means±SEM. For studies comparing 2 groups,the Student's t test was used, while for studies comparing more than 2groups, one- or two-way ANOVA with the appropriate post hoc testing wasused, with * p≤0.05, ** p≤0.01, *** p≤0.001, and **** p≤0.0001.

Methods for the Ly95-RISR-RIAD

A construct was made fusing the RISR-RIAD construct to the Ly95transgenic TCR. This was inserted into a lentivirus and used totransduce T cells using the standard approach.

T cells were evaluated by FACS to confirm the transfection efficiency ofthe CAR. The T cells were then exposed at three different T cell totumor ratios to A549 cells expressing HLA-2 and NYESO. Their ability tokill the tumor cells was assessed. The efficacy of the Ly95 vsLy95-RISR-RIAD cells were compared.

To study the ability of RISR-RIAD to prevent immunosuppression, the Tcells were then exposed at three different T cell to tumor ratios toA549 cells expressing HLA-2 and NYESO. This was done with normal mediaor with different concentrations of adenosine or PGE2. Their ability tokill the tumor cells over an 18 hrs period was assessed. The efficacy ofthe Ly95 vs Ly95-RISR-RIAD cells were compared.

Supernatants were collected from each well and used an ELISA to measureInterferon-gamma as a measure of T cell activation.

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

1. A composition comprising a nucleic acid molecule encoding a chimericantigen receptor and a nucleic acid molecule encoding a RIAD polypeptideor an Ezrin polypeptide.
 2. The composition of claim 1, wherein saidRIAD polypeptide comprises a RISR subunit or an Ezrin-derived RISRsubunit.
 3. The composition of claim 2, wherein said RIAD polypeptidecomprises a RISR subunit and said RISR subunit comprises a polypeptidehaving an amino acid sequence having at least 90% identity to SEQ ID NO:64.
 4. The composition of claim 3, wherein said RISR subunit comprises apolypeptide having an amino acid sequence having at least 95% identityto SEQ ID NO:
 64. 5. The composition of claim 4, wherein said RISRsubunit comprises a polypeptide having an amino acid sequence having SEQID NO:
 64. 6. The composition of claim 1, wherein said RIAD polypeptidecomprises an amino acid sequence having at least 90% identity to thesequence of SEQ ID NO:
 63. 7. The composition of claim 6, wherein saidRIAD polypeptide comprises an amino acid sequence having at least 95%identity to the sequence of SEQ ID NO:
 63. 8. The composition of claim7, wherein said RIAD polypeptide comprises an amino acid sequence havingthe sequence of SEQ ID NO:
 63. 9. The composition of claim 2, whereinsaid RIAD polypeptide comprises an amino acid sequence having thesequence of SEQ ID NO:
 65. 10. The composition of claim 1, wherein saidchimeric antigen receptor comprises an antigen binding domain, atransmembrane domain, and an intracellular domain comprising acostimulatory domain and/or a primary signaling domain.
 11. Thecomposition of claim 10, wherein said antigen binding domain binds atumor antigen.
 12. The composition of claim 11, wherein said tumorantigen is selected from the group consisting of: CD19; CD123; CD22;CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7,CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1);CD33; epidermal growth factor receptor variant III (EGFRvIII);ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcaSer/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y) antigen; CD24; Platelet derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp 100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NYBR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ES0-1); Cancer/testis antigen 2 (LAGE-1a); Melanomaassociated antigen 1 (MAGE-A1); ETS translocation-variant gene 6,located on chromosome 12p (ETV6-AML); sperm protein 17 (SP A1 7); XAntigen Family, Member IA (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-I (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fas-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-I (PCT A-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); NAcetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B 1(CYPIB 1); CCCTC-Binding Factor (Zinc Finger Protein)Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (L Y75); Glypican-3(GPC3); Fe receptor-like 5 (FCRL5); and immunoglobulin lambda-likepolypeptide 1 (IGLL1).
 13. The composition of claim 10, wherein saidintracellular domain comprises a primary signaling domain and acostimulatory domain.
 14. The composition of claim 10, wherein saidintracellular domain comprises a primary signaling domain comprising afunctional signaling domain of one or more proteins selected from thegroup consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, commonFcR gamma (FCER1G), FcR beta (Fc Epsilon R1b), CD79a, CD79b, FcgammaRIIa, DAP10, and DAP12.
 15. The composition of claim 10, wherein saidintracellular domain comprises a costimulatory domains comprising afunctional domains of one or more proteins selected from the groupconsisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1,GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.
 16. The composition ofclaim 10, wherein the antigen binding domain comprises an antibody, anantibody fragment, an scFv, a Fv, a Fab, a (Fab′)2, a single domainantibody (SDAB), a VH or VL domain, or a camelid VHH domain.
 17. Thecomposition of claim 10, wherein the transmembrane domain comprises atransmembrane domain of a protein selected from the group consisting ofthe alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS(CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103,ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, andNKG2C.
 18. The composition of claim 10, wherein the encodedtransmembrane domain comprises: an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications of an amino acid sequence of SEQ ID NO: 12, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO: 12; or theamino acid sequence of SEQ ID NO:
 12. 19. The composition of claim 10,wherein the nucleic acid sequence encoding the transmembrane domaincomprises a nucleotide sequence of SEQ ID NO: 13, or a sequence with95-99% identity thereof.
 20. The composition of claim 10, wherein theantigen binding domain is connected to the transmembrane domain by ahinge region, wherein said hinge region nucleic acid encodes an aminoacid sequence comprising SEQ ID NO: 6, or a sequence with 95-99%identity thereof; or said hinge region comprises the nucleotide sequenceof SEQ ID NO: 7, or a nucleotide sequence with 95-99% identity thereof;or wherein the chimeric antigen receptor comprises a leader region,wherein said leader region encodes an amino acid sequence comprising SEQID NO: 2, or a sequence with 95-99% identity thereof; or said leaderregion comprises the nucleotide sequence of SEQ ID NO: 3, or anucleotide sequence with 95-99% identity thereof.
 21. The composition ofclaim 10, wherein the encoded intracellular domain comprises thesequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ IDNO: 18 or SEQ ID NO: 20, wherein the sequences comprising theintracellular signaling domain are expressed in the same frame and as asingle polypeptide chain.
 22. The composition of claim 10, wherein thenucleic acid sequence encoding the intracellular signaling domaincomprises a sequence of SEQ ID NO:15 or SEQ ID NO: 17, or a sequencewith 95-99% identity thereof, and a sequence of SEQ ID NO: 19 or SEQ IDNO:21, or a sequence with 95-99% identity thereof.
 23. The compositionof claim 1, wherein the encoded chimeric antigen receptor furthercomprises a leader sequence comprising SEQ ID NO:
 2. 24. The compositionof claim 1, wherein said chimeric antigen receptor and RIAD polypeptideare encoded by a single nucleic molecule in the same frame and as asingle polypeptide chain.
 25. The composition of claim 24, wherein saidRIAD polypeptide is attached to the N-terminus of said chimeric antigenreceptor.
 26. The composition of claim 24, wherein said RIAD polypeptideand chimeric antigen receptor are separated by one or more peptidecleavage sites.
 27. The composition of claim 26, wherein said peptidecleavage site is an autocleavage site or a substrate for anintracellular protease.
 28. The composition of claim 27, wherein saidpeptide cleavage site is a T2A site.
 29. The composition of claim 1,wherein said chimeric antigen receptor and said RIAD polypeptide areencoded by a single nucleic molecule and are not expressed as a singlepolypeptide.
 30. The composition of claim 29, wherein the expression ofsaid chimeric antigen receptor and said RIAD polypeptide is controlledby a common promoter.
 31. The composition of claim 30, wherein thenucleic acid encoding said chimeric antigen receptor and the nucleicacid encoding said RIAD polypeptide are separated by an internalribosomal entry site.
 32. The composition of claim 29, wherein theexpression of said chimeric antigen receptor and said RIAD polypeptideis controlled by separate promoters.
 33. The composition of claim 1,wherein said composition consists of a single isolated nucleic acid. 34.A polypeptide encoded by the single nucleic acid molecule of claim 24.35. A vector comprising the single nucleic acid molecule of claim 24,wherein the vector is selected from the group consisting of a DNAvector, an RNA vector, a plasmid, a lentivirus vector, adenoviralvector, or a retrovirus vector.
 36. The vector of claim 35, furthercomprising a promoter chosen from an EF-1 promoter, a CMV IE genepromoter, an EF-1α promoter, an ubiquitin C promoter, or aphosphoglycerate kinase (PGK) promoter.
 37. The vector of claim 36,wherein the EF-1 promoter comprises a sequence of SEQ ID NO:
 1. 38. Thevector of claim 35, wherein the vector is an in vitro transcribedvector, or the vector further comprises a poly(A) tail or a 3′UTR. 39.The composition of claim 1, wherein said nucleic acid molecule encodinga chimeric antigen receptor and nucleic acid molecule encoding an RIADpolypeptide are separate nucleic acid molecules.
 40. An immune effectorcell comprising the nucleic acid molecule encoding a chimeric antigenreceptor and nucleic acid molecule encoding a RIAD polypeptide or Ezrinpolypeptide of claim
 1. 41. The cell of claim 40, wherein the immuneeffector cell is a human T cell or a human NK cell, optionally, whereinthe T cell is diacylglycerol kinase (DGK) and/or Ikaros deficient.
 42. Amethod of making a chimeric antigen receptor and RIADpolypeptide-expressing or Ezrin polypeptide-expressing immune effectorcell, comprising introducing a vector of claim 35, into an immuneeffector cell, under conditions such that the chimeric antigen receptormolecule is expressed.
 43. The method of claim 42, further comprising:providing a population of immune effector cells; and removing Tregulatory cells from the population, thereby providing a population ofT regulatory-depleted cells; wherein steps a) and b) are performed priorto introducing the nucleic acid (or nucleic acids) encoding the chimericantigen receptor and RIAD polypeptide or Ezrin polypeptide to thepopulation.
 44. The method of claim 43, wherein the T regulatory cellsare removed from the cell population using an anti-CD25 antibody, or ananti-GITR antibody.
 45. A method of providing anti-tumor immunity in asubject comprising administering to the subject an effective amount ofthe immune effector cell of claim 39, wherein the cell is an autologousT cell or an allogeneic T cell, or an autologous T cell or an allogeneicNK cell.
 46. The method of claim 45, wherein the allogeneic T cell orallogeneic NK cell lacks expression or has low expression of afunctional TCR or a functional HLA.
 47. A method of treating a subjecthaving a disease associated with expression of a tumor antigen,comprising administering to the subject an effective amount of an immuneeffector cell of claim 40, thereby treating the subject.
 48. The methodof claim 47, said method further comprising administering an agent thatincreases the efficacy of the immune effector cell, thereby treating thesubject.
 49. The method of claim 48, wherein said agent is chosen fromone or more of: a protein phosphatase inhibitor; a kinase inhibitor; acytokine; an inhibitor of an immune inhibitory molecule; or an agentthat decreases the level or activity of a TREG cell.
 50. The method ofclaim 47, wherein the disease associated with expression of the tumorantigen is selected from the group consisting of a proliferativedisease, a precancerous condition, a cancer, and a non-cancer relatedindication associated with expression of the tumor antigen.
 51. Themethod of claim 50, wherein the cancer is a hematologic cancer chosenfrom one or more of chronic lymphocytic leukemia (CLL), acute leukemias,acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL),T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia(CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendriticcell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma,follicular lymphoma, hairy cell leukemia, small cell- or a largecell-follicular lymphoma, malignant lymphoproliferative conditions, MALTlymphoma, mantle cell lymphoma, marginal zone lymphoma, multiplemyeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin'slymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoiddendritic cell neoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.52. The method of claim 51, wherein the cancer is selected from thegroup consisting of colon cancer, rectal cancer, renal-cell carcinoma,liver cancer, non-small cell carcinoma of the lung, cancer of the smallintestine, cancer of the esophagus, melanoma, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, solid tumors of childhood, cancer ofthe bladder, cancer of the kidney or ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, T-cell lymphoma, environmentally induced cancers, combinationsof said cancers, and metastatic lesions of said cancers. 53.-54.(canceled)